Protease Enzyme and Uses Thereof

ABSTRACT

The present invention is related to a fungal serine protease enzyme, which said enzyme has serine protease activity and comprises an amino acid sequence of  Malbranchea  ALKO4122 mature protease as defined in SEQ ID NO:18 or an amino acid sequence having at least 66% identity to the amino acid sequence of SEQ ID NO:18. Also disclosed is an isolated nucleic acid molecule, comprising a polynucleotide sequence which encodes a fungal serine protease enzyme, nucleic acid sequences encoding said protease, a host cell and a process of producing a polypeptide having serine protease activity. Said protease is useful as an enzyme preparation applicable in detergent compositions and for treating fibers, wool, hair, leather, or silk, for treating food or feed, or for any applications involving modification, degradation or removal of proteinaceous material.

CROSS REFERENCE TO RELATED APPLICATIONS

Under 35 U.S.C. §119(e), this application claims the benefit of bothU.S. Provisional Application No. 61/470,168, filed Mar. 31, 2011, andFinnish Patent Application No. 20115310, filed Mar. 31, 2011. Thecontents of each of the foregoing applications are hereby incorporatedby reference in their entireties in this application.

FIELD OF THE INVENTION

The present invention relates to a serine protease enzyme, particularlyto a fungal serine protease enzyme useful in various applications,particularly in detergents. The invention relates to a nucleic acidmolecule encoding said enzyme, a recombinant vector, a host cell forproducing said enzyme, an enzyme composition comprising said enzyme aswell as a process for preparing such composition. This invention relatesalso to various uses of said enzyme and compositions comprising saidenzyme.

BACKGROUND

Microbial proteases are among the most important hydrolytic enzymes andfind applications in various industrial sectors, such as detergents,food, leather, pharmaceuticals, diagnostics, waste management and silverrecovery. Microbial extracellular proteases account for a major part ofthe total worldwide industrial enzyme sales (Cherry and Fidantsef,2003). Approximately 90% of the commercial proteases are detergentenzymes (Gupta et al., 2002). The commercial detergent preparationscurrently in use comprise the naturally occurring alkaline serineproteases (EC 3.4.21) of the subtilisin family or subtilisins (EC3.4.21.62), originating from Bacillus species, or are recombinantprotease preparations thereof (Maurer, 2004).

Examples of commercial proteases are such as Subtilisin Carlsberg(Alcalase®), Subtilisin 309 (Savinase®), Subtilisin 147 (Esperase®),Kannase®, Everlase®, Ovozyme®, and the cold-wash protease Polarzyme®(Novozymes A/S, DK), Purafect®, Purafect® Ox, Purafect® Prime andProperase® (Genencor Int., Inc., USA), and the BLAP S and X series(Henkel, D E).

Several alkaline serine proteases and genes encoding these enzymes havealso been isolated from eukaryotic organisms, including yeast andfilamentous fungi. U.S. Pat. No. 3,652,399 and EP 519229 (TakedaChemical Industries, Ltd., JP) disclose an alkaline protease from thegenus Fusarium (asexual state, teleomorph) or Gibberella, (sexual state,anamorph) particularly from Fusarium sp. S-19-5 (ATCC 20192, IFO 8884),F. oxysporum f. sp. lint (IFO 5880) or G. saubinetti (ATCC 20193,IF06608), useful in the formulation of detergent and other cleansercompositions. WO1994025583 (NovoNordisk A/S, DK) discloses an activetrypsin-like protease enzyme derivable from a Fusarium species, inparticular a strain of F. oxysporum (DSM 2672), and the DNA sequenceencoding the same. The amino acid and nucleotide sequences of the serineproteases from F. equiseti and F. acuminatum have been disclosed in WO2010125174 and WO 2010125175, respectively (AB Enzymes Oy, FI). Also,alkaline proteases from fungal species such as Tritirachium andConidiobolus have been reported (reviewed in Anwar and Saleemuddin 1998)

The major problem in the use of proteases in liquid detergents is theirinstability. In liquid detergents enzymes are in direct contact withwater and chatropic agents like anionic surfactants and complexingagents, which can lead to irreversible denaturation. Proteases degradeproteins including other enzymes in detergent formulations andthemselves. The auto-proteolysis is enhanced by surfactants and heat.Thus, the stability of liquid detergents containing protease representsa major challenge for product development (Maurer, 2010).

Various methods have been used for improving the stability of industrialserine proteases. WO 92/03529 (NovoNordisk A/S, DK), US 2009/096916(Genencor Int. Inc., US) and WO 2007/145963 (Procter & Gamble Co., US)disclose the use of a reversible protease inhibitor of a peptide orprotein type. Liquid detergent compositions comprising proteases ofteninclude protease inhibitors such as boric acid with or without polyolsto inhibit the activity of proteases. One example of such inhibitors is4-formyl phenyl boronic acid (4-FPBA) disclosed in US 2010/0120649(Novozymes A/S, DK). The stability of proteases has also been improvedby using a combination of halide salts with polyols (WO 02/08398,Genencor Int. Inc., US). EP 0352244A2 (NovoNordisk A/S, DK) suggestsimproving the stability of Bacillus derived enzymes using amphotericcompounds, such as surfactants.

Based on the information derived from the crystal structures andsequence similarity comparisons between homologous proteins, variantswith improved stability and/or improved performance may be designed.Variants of the natural serine proteases with improved catalyticefficiency and/or better stability towards temperature, oxidizing agentsand different washing conditions, as well as improved storage stabilityin liquid detergents have been developed through site-directed and/orrandom mutagenesis.

Thermomycolin EC 3.4.21.65, isolated as an extracellular alkalineendopeptidase, is produced by a thermophilic fungus Malbranchea pulcellavar. sulfurea. Thermomycolin is described as a 325 residue, single-chainprotein. It has the active-site sequence -Leu-Ser-(Gly)-Thr-Ser*-Met-,which is typical for a member of the subtilisin family. Thermomycolinpossesses one disulfide bridge, which is exceptional. Thermomycolin isnot as thermostable as the extracellular serine proteinases ofthermophilic bacteria, but it is more stable than most fungalproteinases (Gaucher and Stevenson, 2004). According to Ong and Gaucher(1975) the thermal inactivation of thermomycolin occurs at 73° C. in thepresence of 10 mM Ca²⁺. Thermomycolin hydrolyses casein on broad pHrange. The optimum pH for hydrolysis of casein is about 8.5.

Abu-Shady et al. (2001) disclose properties of a protease fromMalbranchea sulfurea that is a local isolate from soil samples collectedfrom butcheries in Egypt and cultured to obtain a protease enzyme. Thispublication describes the relative activity of M. sulphurea protease inthe presence of certain detergents at low concentrations (0.7%) at30-90° C. using preincubation time of 15-60 mM, i.e. at conditionsresembling washing conditions. However, it does not give any indicationof the stain removal performance or the storage stability of thisprotease in detergent itself, which are essential properties for thesuitability for use of a protease in detergent formulations. Thepublication also describes the temperature and pH profiles of apartially purified protease. The optimum temperature of the protease isat 50° C. and optimum pH at 9.0.

Despite the fact that numerous patent publications, reviews and articleshave been published, disclosing fungal serine proteases from variousmicroorganisms, for example, the low temperature alkaline proteases fromactinomycete (Nocardiopsis dassonvillei) and fungal (Paecilomycesmarquandii) microorganisms, e.g. in EP 0290567 and EP 0290569 (NovoNordisk A/S, DK), there is still a great need for new proteases, whichare suitable for and effective in modifying, degrading and removingproteinaceous materials of different stains, particularly at low ormoderate temperature ranges and which are stable in the presence ofdetergents with highly varying properties. Due to the autocatalyticproperty of serine proteases, the stability during storage is also veryimportant.

It is also desirable that the serine protease can be produced in highamounts, and can be cost-effectively down-stream processed, by easyseparation from fermentation broth and mycelia.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a serine protease offungal origin which is active at broad pH ranges and functionsparticularly well at low and moderate temperatures. The serine proteasesfor detergent application have to be stable also in the presence ofdetergents and to be compatible with detergents. Particularly, theobject of the invention is to provide a serine protease, which iscapable of removing proteinaceous material, including stains in washinglaundry and dishes, at lower temperatures than a commercial enzymepreparation, whereby for example more sensitive materials can be washedand energy is saved. Further objects of the invention are to provide anucleic acid molecule encoding said enzyme, a recombinant vector, a hostcell for producing said enzyme, a composition comprising said enzyme, aprocess for preparing such composition, as well as s uses of said enzymeand compositions comprising said enzyme.

The fungal serine protease according to the invention can be produced inhigh-yielding fungal hosts and its down-stream processing, e.g.separation of fermentation broth and mycelia is easy to perform.

The present invention relates to a fungal serine protease enzyme, whichhas serine protease activity and comprises an amino acid sequence havingat least 66% identity to the amino acid sequence as defined in SEQ IDNo:18. A preferred serine protease is derived from Malbrancheacinnamomea (Lib.) Oorschot de Hoog.

In the present context, “derived from” is intended not only to indicatea serine protease produced or producible by a strain of the organism inquestion, but also a serine protease encoded by a DNA sequence isolatedfrom such strain and produced in a host organism transformed with saidDNA sequence. Finally, the term is intended to indicate a serineprotease, which is encoded by a DNA sequence of synthetic and/or cDNAorigin and which has the identifying characteristics of serine proteasein question.

Preferably the invention relates to a fungal serine protease enzyme,which has serine protease activity and comprises an amino acid sequencehaving at least 66% identity to the amino acid sequence of the matureMalbranchea ALKO4122 protease as defined in SEQ ID No:18 or the an aminoacid sequence of the mature Malbranchea ALKO4122 protease as defined inSEQ ID No:18.

The fungal serine protease enzyme of the invention is obtainable fromMalbranchea, preferably from Malbranchea cinnamomea (Lib.) Oorschot deHoog (Synonym of Malbranchea pulchella var. sulfurea (Miehe) Cooney & R.Emers.). According to the particularly preferred embodiment the serineprotease enzyme of the invention is obtainable from the MalbrancheaALKO4122 strain deposited under accession number CBS 128533 or from thestrain Malbranchea ALKO4178 deposited under accession number CBS 128564.The protease enzyme of Malbranchea ALKO4178 is essentially identicalwith the protease enzyme of Malbranchea ALKO4122 strain.

The fungal serine protease enzyme has a molecular mass between 20 and 35kDa. The temperature optimum of the enzyme is in the range from 30° C.to 80° C. at pH 8.5, preferably at approximately 70° C. The pH optimumof the enzyme is in the range from at least pH 6 to pH 10 at 50° C.,preferably at pH 10. The temperature and pH characteristics weredetermined using 30 min reaction time and casein as a substrate.

The fungal serine protease of the invention is capable in degrading orremoving proteinaceous stains in the presence of detergents at atemperature from 0° C. to 90° C., preferably at a temperature from 5° C.to 60° C., particularly preferably at a temperature from 10° C. to 40°C.

The fungal serine protease enzyme of the invention is encoded by anisolated polynucleotide sequence, which hybridizes under stringentconditions to a polynucleotide sequence included in plasmid pALK3092comprising the nucleotide sequence SEQ ID No:11, deposited in E. coliRF8758 under accession number DSM 24426 or to the sequence encoding themature ALKO4122 protease SEQ ID No:17. Alternatively, the fungal serineprotease enzyme of the invention is encoded by an isolatedpolynucleotide sequence, which hybridizes under stringent conditions toa polynucleotide sequence included in plasmid pALK3093 comprising thenucleotide sequence SEQ ID No:12, deposited in E. coli RF8759 underaccession number DSM 24427.

Said enzyme is encoded by an isolated polynucleotide sequence, whichencodes a polypeptide comprising an amino acid sequence of the matureMalbranchea ALKO4122 protease as defined in SEQ ID No:18 or an aminoacid sequence having at least 66% identity to the amino acid sequence ofthe mature Malbranchea ALKO4122 protease as defined in SEQ ID No:18.Preferably, said enzyme is encoded by an isolated nucleic acid moleculecomprising the nucleotide sequence SEQ ID No:17.

The full-length fungal serine protease enzyme of the invention isencoded by the polynucleotide sequence included in pALK3094 deposited inEscherichia coli RF8791 under accession number DSM 24410.

The fungal serine protease enzyme is produced from a recombinantexpression vector comprising the nucleic acid molecule encoding a fungalserine protease of the invention, operably linked to regulatorysequences capable of directing the expression of the serine proteaseenzyme in a suitable host. Suitable hosts include heterologous hosts,preferably microbial hosts of the genus Trichoderma, Aspergillus,Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Penicillium,Myceliophthora and Mortiriella. Preferably said enzyme is produced inTrichoderma or Aspergillus, most preferably in T. reesei.

The present invention relates also to an isolated nucleic acid moleculecomprising a polynucleotide sequence encoding a serine protease enzymeselected from the group consisting of:

-   -   (a) a nucleic acid molecule encoding a polypeptide having serine        protease activity and comprising the amino acid sequence as        depicted in SEQ ID No:18;    -   (b) a nucleic acid molecule encoding a polypeptide having serine        protease activity and at least 66% identity to the amino acid        sequence of SEQ ID No:18;    -   (c) a nucleic acid molecule comprising the coding sequence of        the nucleotide sequence as depicted in SEQ ID No: 17;    -   (d) a nucleic acid molecule comprising the coding sequence of        the polynucleotide sequence contained in DSM 24410;    -   (e) a nucleic acid molecule the coding sequence of which differs        from the coding sequence of a nucleic acid molecule of any one        of (c) to (d) due to the degeneracy of the genetic code; and    -   (f) a nucleic acid molecule hybridizing under stringent        conditions to a nucleic acid molecule contained in DSM 24426, or        SEQ ID No:17 encoding a polypeptide having serine protease        activity and an amino acid sequence which shows at least 66%        identity to the amino acid sequence as depicted in SEQ ID No:18.

The invention further relates to a recombinant expression vectorcomprising the nucleotide sequence of the invention operably linked toregulatory sequences capable of directing expression of said serineprotease gene in a suitable host. Suitable hosts include heterologoushosts, preferably microbial hosts of the genus Trichoderma, Aspergillus,Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus, Myceliophthoraand Mortiriella. Preferably said enzyme is produced in Trichoderma orAspergillus, most preferably in T. reesei.

The invention relates also to a host cell comprising the recombinantexpression vector as described above. Preferably, the host cell is amicrobial host, such as a filamentous fungus. Preferred hosts are of agenus Trichoderma, Aspergillus, Fusarium, Humicola, Chrysosporium,Neurospora, Rhizopus, Penicillium, Myceliophthora and Mortiriella. Morepreferably the host is Trichoderma or Aspergillus, most preferably afilamentous fungus T. reesei.

The present invention relates to a process of producing a polypeptidehaving serine protease activity, said process comprising the steps ofculturing the host cell of the invention and recovering the polypeptide.Also within the invention is a polypeptide having serine proteaseactivity encoded by the nucleic acid sequence of the invention and whichis obtainable by the process described above.

The invention relates to a process for obtaining an enzyme preparationcomprising the steps of culturing a host cell of the invention andeither recovering the polypeptide from the cells or separating the cellsfrom the culture medium and obtaining the supernatant. Within theinvention is also an enzyme preparation obtainable by the processdescribed above.

The invention relates to an enzyme preparation, which comprises theserine protease enzyme of the invention.

The invention further relates to a composition comprising the serineprotease enzyme of the invention.

The enzyme preparation or composition (e.g. detergent formulation)containing the protease enzyme of the invention may further compriseother enzymes selected from the group consisting of proteases (otherprotease than that of the invention), amylases, cellulases, lipases,xylanases, mannanases, cutinases, pectinases and oxidases with orwithout a mediator as well as suitable additives selected from the groupconsisting of stabilizers, buffers, surfactants, bleaching agents,mediators, anti-corrosion agents, builders, anti-redeposition agents,optical brighteners, dyes, pigments, perfumes, caustics, abrasives andpreservatives, etc.

The spent culture medium of the production host can be used as such, orthe host cells may be removed, and/or it may be concentrated, filtratedor fractionated. It may also be dried. The enzyme preparation and thecomposition comprising the serine protease enzyme of the invention maybe in the form of liquid, powder, granulate or tablets. The enzyme maybe in immobolized form in the preparation or in the composition.

Also within the invention is the use of the serine protease enzyme orthe enzyme preparation of the invention for detergents, for treatingfibers, for treating proteinaceous material such as wool, hair, leather,silk, for treating food or feed, or for any applications involvingmodification, degradation or removal of proteinaceous material.Particularly, the enzyme or enzyme preparation is useful as a detergentadditive in detergent liquids, detergent powders and detergent tablets.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 (1A and 1B) shows the nucleotide sequence of the MalbrancheaALKO4122 protease gene, its partial promoter (50 nucleotides upstreamfrom ATG) and terminator sequences (60 nucleotides downstream from thestop codon) and the deduced amino acid sequence of the encoded protease.The putative signal peptide, analyzed by SignalP V3.0 program is inlower case letters and underlined. The pro sequence and the deducedamino acids of the pro sequence are in lower case letters. The maturenucleotide and peptide sequences are in capital letters. The threeputative intron sequences are in lower case, italic letters and markedby a dotted line below the nucleotide sequence. The stop codon is shownby an asterisk below the sequence. The locations of primers DET27(5′-sense primer, s) and DET28 (3′-antisense primer, as) used in PCRcloning of the Malbranchea ALKO4178 protease gene are underlined usingdouble lines.

FIG. 1A shows the nucleotide sequence of the Malbranchea ALKO4122protease gene and its partial promoter. The protease gene sequenceincludes nucleotides from 51 to 960, the sequence region encoding theamino acid sequence from Met 1 to Val 279 and the first codon of Ala 280of the protease.

FIG. 1B shows the nucleotide sequence of the Malbranchea ALKO4122protease gene and its partial terminator. The protease gene sequenceincludes nucleotides from 961 to 1486 (the stop codon TAA included), thesequence region encoding the amino acid sequence from Ala 280 (the twolast codons) to Arg 401 of the protease.

FIG. 2 schematically shows the map of pALK2777 plasmid used as abackbone for constructing the pALK3097 expression cassette. TheMalbranchea protease gene was ligated between the cbh1 (cel7A) promoter(exact fusion via the SacII site) and terminator sequences (via theBamHI site in the linker) into SacII-BamHI cleaved pALK2777. For furtherdetails, see Example 2. The pALK2777 plasmid includes a synthetic amdSmarker gene for transformant screening. pcbh1, cbh1 promoter; tcbh1,cbh1 terminator; s_amdS, synthetic amdS marker gene (cDNA); Linker,linker sequence including e.g. BamHI site.

FIG. 3 schematically shows the pALK3097 cassette isolated from thevector backbone by Nod digestion and used for expressing the MalbrancheaALKO4122 protease gene in Trichoderma reesei. pcbh1, cbh1 promoter;tcbh1, cbh1 terminator; s_amdS, synthetic amdS marker gene (cDNA);Linker, linker sequence.

FIG. 4 shows the partially purified recombinant protein analysed on 12%SDS PAGE gel. Lane 1. Sample of the partially purified Malbrancheaprotease, Lane 2. MW marker (Page Ruler Unstained Protein Ladder,Fermentas).

FIG. 5 (5A and 5B) shows the relative activity of the enzyme atdifferent temperatures and pH values.

FIG. 5A describes the temperature profile of the recombinant Malbrancheaprotease and Savinase® 16 L assayed at pH 8.5 using 30 min reaction timeand casein as a substrate. The data points are averages of threeseparate measurements.

FIG. 5B describes the effect of pH on the activity of recombinantMalbrachea protease and Savinase® 16 L. The buffer used was 40 mMBritton-Robinson buffer, casein was used as a substrate, reaction timewas 30 min and reaction temperature was 50° C. The data points areaverages of three separate measurements

FIG. 6 (6A, 6B, 6C and 6D) describes the stain removal performance ofrecombinant Malbranchea ALKO4122 protease with blood/milk/ink stain(Art. 117, CO+PES, Serial No. 11-08, new batch, EMPA) at 10-50° C.,approx. pH 8, 60 min in the presence of Commercial liquid detergent withconcentration of 5 g/l. Commercial protease preparations Savinase® 16 Land Savinaseg Ultra 16 L were used for comparison.

FIG. 6A describes the stain removal performance at 10° C.,

FIG. 6B describes the stain removal performance at 20° C.

FIG. 6C describes the stain removal performance at 30° C.

FIG. 6D describes the stain removal performance at 50° C.

FIG. 7 (7A, 7B, 7D and 7C) describes the stain removal performance ofrecombinant Malbranchea ALKO4122 protease with blood/milk/ink stain(Art. 117, CO+PES, Serial No. 10-07, old batch, EMPA) at 10-50° C.,approx. pH 8, 60 min in the presence of Commercial liquid detergent withconcentration of 5 g/l. Savinase® Ultra 16 L was used for comparison.

FIG. 7A describes the stain removal performance at 10° C.

FIG. 7B describes the stain removal performance at 20° C.

FIG. 7C describes the stain removal performance at 30° C.

FIG. 7D describes the stain removal performance at 50° C.

FIG. 8 (8A, 8B, 8C and 8D) describes the stain removal performance ofrecombinant Malbranchea ALKO4122 protease with blood/milk/ink stain(Art. 117, CO+PES, Serial No. 11-08, new batch, EMPA) at 10-50° C.,approx. pH 8, 60 min in the presence of Commercial liquid detergent withconcentration of 5 g/l. Savinase® 16 L and Savinase® Ultra 16 L wereused for comparison.

FIG. 8A describes the stain removal performance at 10° C. (enzyme dosagecalculated as protein).

FIG. 8B describes the stain removal performance at 20° C. (enzyme dosagecalculated as protein).

FIG. 8C describes the stain removal performance at 30° C. (enzyme dosagecalculated as protein).

FIG. 8D describes the stain removal performance at 50° C. (enzyme dosagecalculated as protein).

FIG. 9 (9A, 9B, 9C, 9D, 9E, 9F, 9G and 9H) shows the performance ofrecombinant Malbranchea ALKO4122 protease on different stains in LaunderOmeter tests in the presence of Commercial liquid detergent withconcentration of 5 g/l at 30 and 60° C., 60 min, approx. pH 8.Commercial preparation Savinase® Ultra 16 L was used for comparison.

FIG. 9A shows performance on Grass, Cotton, Art. 164 (Serial No. 23-03),EMPA at 30° C.

FIG. 9B shows performance on Grass, Cotton, Art. 164 (Serial No. 23-03),EMPA at 60° C.

FIG. 9C shows performance on Blood/milk/ink, Cotton, Art. 116 (SerialNo. 18-16), EMPA at 30° C.

FIG. 9D shows performance on Blood/milk/ink, Cotton, Art. 116 (SerialNo. 18-16), EMPA at 60° C.

FIG. 9E shows performance on Blood/milk/ink, CO+PES, Art. 117 (SerialNo. 11-08, new batch), EMPA at 30° C.

FIG. 9F shows performance on Blood/milk/ink, CO+PES, Art. 117 (SerialNo. 11-08, new batch), EMPA at 60° C.

FIG. 9G shows performance on Blood/milk/ink, CO+PES, Art. 117 (SerialNo. 10-07, old batch), EMPA at 30° C.

FIG. 9H shows performance on Blood/milk/ink, CO+PES, Art, 117 (SerialNo. 10-07, old batch), EMPA at 60° C.

FIG. 10 (10A and 10B) shows the performance of the protease in thepresence of detergent powder.

FIG. 10A describes the performance of recombinant Malbranchea ALKO4122protease on Blood/milk/ink, CO+PES, Art. 117 (Serial No. 11-08), EMPA inthe presence of Commercial traditional detergent powder (described inExample 8) 5 g/l at 50° C., 60 min, pH approx. 10.5. Savinase® Ultra 16L was used for comparison.

FIG. 10B describes the performance of recombinant Malbranchea ALKO4122protease on Blood/milk/ink, CO+PES, Art. 117 (Serial No. 11-08), EMPA inthe presence of detergent powder Art. 601, EMPA 5 g/1, 50° C., 60 min,pH approx. 10. Savinase® Ultra 16 L was used for comparison.

FIG. 11 (11A and 11B) shows the stability of the protease during storageat 37° C.

FIG. 11A shows the stability of recombinant Malbranchea ALKO4122protease during storage at 37° C., when preserved/stabilized with ProxelLV or propylene glycol (PG).

FIG. 11B shows the stability of recombinant protease Fe_RF6318(WO2010125174A1) during storage at 37° C., when preserved/stabilizedwith Proxel LV or propylene glycol.

FIG. 12 (12A and 12B) shows the stability of the protease in detergent.

FIG. 12A shows the stability of recombinant Malbranchea ALKO4122protease in Ecolabel Reference Detergent at 37° C. pH approx. 7.Commercial preparation Savinase® Ultra 16 L and recombinant proteaseFe_RF6318 (WO2010125174A1) were used for comparison. The amount ofenzyme preparation used in detergent was 4% (w/w).

FIG. 12B shows the stability of Malbranchea ALKO4122 protease inCommercial liquid detergent at 37° C. (pH approx. 8). Commercialpreparation Savinase® Ultra 16 L and recombinant protease Fe_RF6318(WO2010125174A1) were used for comparison. The amount of enzymepreparation used in detergent was 4% (w/w).

SEQUENCE LISTING

SEQ ID NO:1 Sequence of DET1 sense primer used in PCR for Malbrancheaprotease probe synthesis.

SEQ ID NO:2 Sequence of DET2 sense primer used in PCR for Malbrancheaprotease probe synthesis.

SEQ ID NO:3 Sequence of DET3 antisense primer used in PCR forMalbranchea protease probe synthesis.

SEQ ID NO:4 Sequence of DET4 antisense primer used in PCR forMalbranchea protease probe synthesis.

SEQ ID NO:5 Sequence of DET5 sense primer used in PCR for Malbrancheaprotease probe synthesis.

SEQ ID NO:6 Sequence of a consensus peptide sequence used for design ofDET1 sense PCR primer.

SEQ ID NO:7 Sequence of a consensus peptide sequence used for design ofDET2 sense PCR primer.

SEQ ID NO:8 Sequence of a consensus peptide sequence used for design ofDET3 antisense PCR primer.

SEQ ID NO:9 Sequence of a consensus peptide sequence used for design ofDET4 antisense PCR primer.

SEQ ID NO:10 Sequence of the peptide sequence used for design of DET5sense PCR primer.

SEQ ID NO:11 Sequence of the PCR fragment obtained using DET5 and DET4in PCR reaction and Malbranchea ALKO4122 genomic DNA as a template. Thisfragment contains a partial Malbranchea protease gene and is an insertin the plasmid pALK3092,

SEQ ID NO:12 Sequence of the PCR fragment obtained using DET5 and DET4in PCR reaction and Malbranchea ALKO4178 genomic DNA as a template. Thisfragment contains a partial Malbranchea protease gene and is an insertin the plasmid pALK3093.

SEQ ID NO:13 The nucleotide sequence encoding the full-length amino acidsequence of Malbranchea ALKO4122 protease. The full-length gene isincluded in the plasmid pALK3094. The protease gene sequence cloned fromMalbranchea ALKO4178 by PCR was identical to this sequence.

SEQ ID NO:14 The full-length amino acid sequence of Malbranchea ALKO4122protease including amino acids Metl to Arg 401 of the full lengthprotease.

SEQ ID NO:15 The nucleotide sequence encoding the amino acid sequence ofthe proenzyme form of Malbranchea ALKO4122 protease.

SEQ ID NO:16 The amino acid sequence of the proenzyme form ofMalbranchea ALKO4122 protease including amino acids Gly 21 to Arg 401 ofthe full length protease.

SEQ ID NO:17 The nucleotide sequence encoding the amino acid sequence ofthe mature form of Malbranchea ALKO4122 protease.

SEQ ID NO:18 The amino acid sequence of the mature form of MalbrancheaALKO4122 protease including amino acids Ala 121 to Arg 401 of the fulllength enzyme.

SEQ ID NO:19 Sequence of the PCR sense primer DET27 used for cloning ofthe Malbranchea ALKO4178 protease gene.

SEQ ID NO:20 Sequence of the PCR antisense primer DET28 used for cloningof the Malbranchea ALKO4178 protease gene.

SEQ ID NO:21 Sequence of the PCR sense primer DET17 used in constructionof the expression cassette in pALK3097.

SEQ ID NO:22 Sequence of the PCR antisense primer DET18 used inconstruction of the expression cassette in pALK3097.

Deposits

Malbranchea ALKO4122 was deposited at the Centraalbureau VoorSchimmelcultures at Uppsalalaan 8, 3508 AD, Utrecht, the Netherlands on20 Dec. 2010 and assigned accession number CBS 128533.

Malbranchea ALKO4178 was deposited at the Centraalbureau VoorSchimmelcultures at Uppsalalaan 8, 3508 AD, Utrecht, the Netherlands on5 Jan. 2011 and assigned accession number CBS 128564.

The E. coli strain RF8791 including the plasmid pALK3094 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 B, D-38124 Braunschweig, Germany on 20 Dec.2010 and assigned accession number DSM 24410.

The E. coli strain RF8758 including the plasmid pALK3092 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 B, D-38124 Braunschweig, Germany on 3 Jan.2010 and assigned accession number DSM 24426.

The E. coli strain RF8759 including the plasmid pALK3093 was depositedat the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ), Inhoffenstrasse 7 B, D-38124 Braunschweig, Germany on 3 Jan.2010 and assigned accession number DSM 24427.

DETAILED DESCRIPTION

The present invention provides a serine protease enzyme of fungalorigin. This protease is active at broad pH ranges and has a widetemperature optimum in washing, and particularly good performance at lowtemperature ranges as well as at moderate and high temperatures. Theenzyme is ideal for detergent applications, withstanding typicaldetergent compositions and being effective at low enzyme levels indetergent solutions. Particularly, the protease is active at applicationtemperatures 0° C.-90° C., the preferred range being from 5° C. to 60°C., more preferably from 10 to 40° C. The protease of the invention isalso highly stable in liquid detergent compositions. Thus, the presentinvention provides a new serine protease for use in detergent and otherapplications, particularly in liquid formulations. The fungal serineprotease can be produced in high-yielding fungal hosts and itsdown-stream processing, e.g. separation of fermentation broth andmycelia is easy to perform.

Particularly, the present invention provides a serine protease enzyme,which has serine protease activity and comprises an amino acid sequencehaving at least 66% identity to the amino acid sequence as defined inSEQ ID No:18. Preferably the present invention provides a fungal serineprotease enzyme, which has serine protease activity and comprises anamino acid sequence as defined in SEQ ID No:18. A preferred serineprotease is derived from Malbranchea cinnamomea (Lib.) Oorschot de Hoog.

By “serine protease” or “serine endopeptidase” or “serineendoproteinase” is in connection to this invention meant an enzymeclassified as EC 3.4.21 by the Nomenclature of the International Unionof Biochemistry and Molecular Biology. Proteases can be classified usinggroup specific inhibitors. The diverse group of serine proteaseinhibitors includes synthetic chemical inhibitors and naturalproteinaceous inhibitors. Thus, the serine protease activity can bedetermined in an assay based on cleavage of a specific substrate or inan assay using any protein containing substrate with or without aspecific inhibitor of serine proteases under suitable conditions.

By the term “serine protease activity” as used in the invention is meanthydrolytic activity on protein containing substrates, e.g. casein,haemoglobin and BSA. The methods for analysing proteolytic activity arewell-known in the literature and are referred e.g. in Gupta et al.(2002).

The serine proteases are synthesized as inactive zymogenic precursors orzymogens in the form of a preproenzyme, which are activated by removalof the signal sequence (secretion signal peptide or prepeptide) and theprosequence (propeptide) to yield an active mature form of the enzyme(Chen and Inouye, 2008). This activation process involves action ofproteases and may result from limited self-digestive or autocatalyticprocessing of the serine protease, e.g. during posttranslational phasesof the production or in the spent culture medium or during the storageof the culture medium or enzyme preparation. Activation of the proenzymemay also be achieved by adding a proteolytic enzyme capable ofconverting the inactive proenzyme into active mature enzyme into theculture medium during or after cultivation of the host organism. Theshortening of the enzyme can also be achieved e.g. by truncating thegene encoding the polypeptide prior to transforming it to the productionhost. The “prepro-form” of the serine protease in the present inventionmeans an enzyme comprising the pre- and propeptides. The “pro-form”means an enzyme, which comprises the propeptide but lacks the prepeptide(signal sequence).

The term “mature” means the form of the serine protease enzyme whichafter removal of the signal sequence (prepeptide) and propeptidecomprises the essential amino acids for enzymatic or catalytic activity.In filamentous fungi it is the native form secreted into the culturemedium. The first amino acid of mature sequence can be determined byN-terminal sequencing of secreted protease. In case no biochemical datais available the location of the N-terminus can be estimated by aligningthe amino acid sequence with mature amino acid sequence(s) of homologousprotein(s). The alignment can be performed using e.g. ClustalW2alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

The largest group of commercial serine proteases are “alkaline serineproteases”, which means that the enzymes are active and stable at pH 9to pH 11 or even at pH 10 to 12.5 (Shimogaki et al., 1991) and haveisoelectric point around pH 9. Determination of the optimal pH of thecatalytic activity can be carried out in a suitable buffer at differentpH values by following the activity on a protein substrate. Typicallythe detergent proteases perform best when the pH value of the detergentsolution in which it works is approximately the same as the pI value forthe enzyme. pI can be determined by isoelectric focusing on animmobilized pH gradient gel composed of polyacrylamide, starch oragarose or by estimating the pI from the amino acid sequence, forexample by using the pI/MW tool at ExPASy server(http://expasy.org/tools/pi_tool.html; Gasteiger et al., 2003).

The molecular masses of mature alkaline serine proteases range between15 and 35 kDa, typically from about 25 to 30 kDa (Rao et al. 1998). Themolecular mass of the serine protease can be determined by massspectrometry or on SDS-PAGE according to Laemmli (1970). The molecularmass can also be predicted from the amino acid sequence of the enzyme.

The temperature optima of most natural serine proteases are around 60°C. (Rao et al., 1998). The temperature optimum of a serine protease canbe determined in a suitable buffer at different temperatures with caseinsubstrate as described in Example 3 or by using other substrates andbuffer systems described in the literature (Gupta et al., 2002).

The mature recombinant Malbranchea serine protease according to theinvention has a molecular weight of approximately 29 kDa, an optimaltemperature of approximately 70° C. at pH 8.5 using 30 min reaction timeand casein as a substrate, and active at alkaline pH range such as pH 10at 50° C. using 30 min reaction time and casein as a substrate. Therecombinant Malbranchea serine protease has a good performance in thepresence of detergents with highly varying properties, at broad, i.e.from low to moderate, and even high temperature ranges. The recombinantMalbranchea serine protease, depending on the washing conditions andauxiliary ingredients and additives in detergents, is usefulparticularly at temperatures at or below 60° C.

To improve the performance of the Malbranchea serine protease in varyingindustrial applications, such as in detergents, it is desirable toimprove the properties of the native enzyme. These properties includee.g. storage stability, stability in the presence or absence ofdetergent, pH stability, oxidative stability or resistance againstbleaching agents and substrate specificity. The autoproteolytic activityof the enzyme has an effect on the storage stability and it should be aslow as possible. It is also self-evident that for example in laundry anddish washing compositions the wash performance of the modified proteaseshould not be impaired in comparison to the parent or precursor proteaseenzyme. In other words it is desirable that the enzyme variants havesimilar or even improved wash performance and stain removal propertieswhen compared to the parent serine protease.

The produced protease enzymes, particularly the serine proteases can bepurified by using conventional methods of enzyme chemistry, such as saltpreparation, ultrafiltration, ion exchange chromatography, affinitychromatography, gel filtration and hydrophobic interactionchromatography. Purification can be monitored by protein determination,enzyme activity assays and by SDS polyacrylamide gel electrophoresis.The enzyme activity and stability of the purified enzyme at varioustemperature and pH values as well as the molecular mass and theisoelectric point can be determined.

The purification of a recombinant serine protease of the presentinvention has been demonstrated in Example 4. The centrifuged andfiltered culture supernatant was applied to HiPrep 26/10 Desaltingcolumn (from GE Healthcare) equilibrated in 20 mM MES pH 5.3. A gelfiltered sample was applied to a 1 mL S Sepharose HP column (from GEHealthcare) equilibrated in 20 mM MES pH 5.3. Proteins were eluted usingincreasing NaCl gradient (0.5 M). Protease containing fractions werepooled and concentrated using Amicon Ultra-4 10,000 CO Centrifugalfilter devices MILLIPORE. A sample was further purified using Superdex75 gel filtration column equilibrated with 20 mM MES, 150 mM NaCl pH5.3. Protease contains fractions were pooled. A final sample wasanalysed on SDS PAGE gel FIG. 4. Naturally, it is possible to separatethe enzyme of the present invention by using other known purificationmethods instead, or in addition to the methods described herein. Therecombinant serine protease was purified as described in Example 4 andused for characterization of pH and temperature profiles as described inExample 5.

Protease activity is generally based on degradation of solublesubstrates. In detergent application proteases have to work onsubstances which are at least partly insoluble. Thus an importantparameter for a detergent protease is the ability to adsorb to andhydrolyse these insoluble fragments.

The serine protease enzyme of the invention may be derived from anyorganism including bacteria, archaea, fungi, yeasts and even highereukaryote, such as plants. Preferably said enzyme originates from afungus, including filamentous fungi and yeasts, for example from a genusselected from the group comprising Malbranchea. Fungal alkalineproteases are advantageous to the bacterial proteases due to the ease ofdown-stream processing to produce a microbe-free enzyme or enzymecomposition. Mycelium can be easily removed through filtrationtechniques prior to the purification of the enzyme.

Mild odor of fungal fermentation products of the present invention is abenefit over Bacillus derived products which typically have anunpleasant odor. Therefore less perfume is needed for the finalcomposition for covering the odor and this makes the product suitablealso for applications where the use of perfumes is not desirable.

The present invention relates to a fungal serine protease, which has agood performance in the presence of detergents with highly varyingproperties, at broad, i.e. from low to moderate temperature ranges from0° C. to 90° C., preferably at temperatures ranging between 5 and 60°C., and particularly preferably at temperatures ranging between 10 and40° C.

In the present invention good performance in presence of detergent meansthat the enzyme, in this case the recombinant fungal serine protease ofthe invention, functions at lower temperature ranges than manycommercial subtilisins. In other words, good performance means that theenzyme is capable of degrading or removing proteinaceous stains ormaterial at low to moderate temperature ranges, but especially at lowertemperature ranges than the present commercial subtilisin products, forexample the commercial subtilisin enzyme product Savinase® orSavinase®Ultra 16 L (Novozymes A/S, DK).

The fungal serine protease of the invention, depending on the washingconditions and auxiliary ingredients and additives in detergents, isuseful particularly in temperatures at or below 60° C. The enzymefunctions also at or below 50° C., at or below 40° C., at or below 30°C. at or below 20° C. and at or below 10° C. It is particularlysurprising that a thermophilic enzyme having a temperature optimumaround 70° C. is effective and useful at temperatures below 40° C., evenat temperatures below 30° C.

In the presence of a detergent, the fungal serine protease of theinvention functions at temperatures as defined above and particularly,said fungal serine protease has a good performance in the presence ofdetergent at or below 40° C. Stain removal performance of the fungalserine protease from Malbranchea in varying test conditions, ondifferent stains, measured as deltaL* is by far better than theperformance of the commercial products, Savinase® and Savinase® Ultra 16L (Novozymes A/S, DK). The results are shown in Examples 6 to 8 and inFIGS. 6 to 10.

According to a preferred embodiment of the invention the recombinantfungal serine protease enzyme is a polypeptide having serine proteaseactivity and comprising an amino acid sequence of the mature MalbrancheaALKO4122 protease as defined in SEQ ID No:18 or an amino acid sequencehaving at least 66% identity to the amino acid sequence of the matureMalbranchea ALKO4122 protease as defined in SEQ ID No:18.

Preferred enzymes show at least 66%, preferably at least 70%, morepreferably at least 75%, even more preferably at least 80% identity.Still more preferably the amino acid sequences show at least 85% or atleast 90% or 95%, more preferably at least 98%, most preferably 99%identity to the amino acid sequence of SEQ ID No:18. Suitably theidentities of the enzymes are compared using the corresponding maturesequence regions.

The serine protease of the present invention in derivable fromMalbranchea, preferably from Malbranchea cinnamomea (Lib.) Oorschot deHoog (Synonym of Malbranchea pulchella var. sulfurea (Miehe) Cooney & R.Emers.), which is a member of EC3.4.21. According to the particularlypreferred embodiment the serine protease enzyme of the invention isderivable from the Malbranchea ALKO4122 strain deposited under accessionnumber CBS 128533 or from the strain Malbranchea ALKO4178 depositedunder accession number CBS 128564. The protease of Malbranchea ALKO4178is essentially identical with the protease of Malbranchea ALKO4122strain.

By the term “identity” is here meant the identity between two amino acidsequences compared to each other within the corresponding sequenceregion having approximately the same amount of amino acids. For example,the identity of a full-length or a mature sequence of the two amino acidsequences may be compared. The amino acid sequences of the two moleculesto be compared may differ in one or more positions, which however doesnot alter the biological function or structure of the molecules. Suchvariation may occur naturally because of different host organisms ormutations in the amino acid sequence or they may be achieved by specificmutagenesis. The variation may result from deletion, substitution,insertion, addition or combination of one or more positions in the aminoacid sequence. The identity of the sequences is measured by usingClustalW2 alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/) withdefault settings (Protein Weight Matrix: Gonnet, Gap open: 10, Gapextension: 0.20, Gap distances 5).

One preferred embodiment of the invention is a mature fungal serineprotease enzyme having serine protease activity and an amino acidsequence of the Malbranchea ALKO4122 protease as defined in SEQ IDNo:18. The mature enzyme lacks the signal sequence or prepeptide and theprosequence or propeptide. The mature serine protease of the inventionincludes amino acids Ala121 to Arg401 of the full length proteasecharacterized in SEQ ID No:14. Thus, within the scope of the inventionis also the full-length Malbranchea ALKO4122 protease enzyme having SEQID No:14 including the signal sequence (prepeptide) and propeptide aswell as the proenzyme form lacking the signal sequence (prepeptide) thushaving SEQ ID No:16.

The present invention relates to a fungal serine protease enzyme, themature form of which has a molecular mass or molecular weight between 20and 35 kDa, preferably between 25 and 33 kDa, more preferably between 28and 30 kDa. The most preferred MW is the predicted molecular mass of 29kDa for the mature polypeptide obtained by using the Compute pI/MW toolat ExPASy server (Gasteiger et al., 2003).

The enzyme of the invention is effective in degrading proteinaceousmaterial at a broad temperature range. The fungal serine protease has atemperature optimum in the range from 30° C. to 80° C. (at least about10% of the maximum activity), preferably from 40° C. to 80° C. (at leastabout 20% of the maximum activity), and more preferably between 50° C.and 80° C. (at least about 40% of the maximum activity), most preferablyfrom 60° C. to 80° C. (at least about 65% of the maximum activity), themaximum activity being at 70° C., when measured at pH 8.5 using 30 minreaction time and casein as a substrate as described in Example 5.

The enzyme has a pH optimum in the range from pH 6 to at least pH 10 at50° C. using 30 min reaction time and casein as a substrate as describedin Example 5. In particular, the pH optimum is between pH 6 and pH 10(at least about 60% of the maximum activity), and more preferablybetween pH 9 and pH 10 (at least about 70% of the maximum activity), andmost preferably at about pH 10.

The serine protease, suitably the fungal serine protease of theinvention has “good performance in the presence of detergent”, i.e. iscapable of degrading or removing proteinaceous stains or material in thepresence of detergent at low temperature ranges, specifically at lowertemperature ranges than the present commercial subtilisin products, forexample the commercial enzyme product Savinase® or Savinase®Ultra 16 L(Novozymes A/S, DK). In the presence of a detergent the enzyme of theinvention functions well between 5° C. and 60° C., preferably at orbelow 50° C. The enzyme functions also in temperatures at or below 40°C., or at or below 30° C.

According to a preferred embodiment of the invention the fungal serineprotease enzyme is encoded by an isolated polynucleotide sequence whichhybridizes under stringent conditions to a polynucleotide or probesequence included in plasmid pALK3092 comprising the nucleotide sequenceSEQ ID No:11 in E. coli RF8758, deposited at the Deutsche Sammlung vonMikroorganismen and Zellkulturen (DSMZ) under accession number DSM24426.

In a similar way, the fungal serine protease enzyme (obtained fromMalbranchea ALKO4178) of the invention is encoded by an isolatedpolynucleotide sequence, which hybridizes under stringent conditions toa polynucleotide sequence included in plasmid pALK3093 comprising thenucleotide sequence SEQ ID No:12, deposited in E. coli RF8759 underaccession number DSM 24427.

Further, the fungal serine protease enzyme of the invention is encodedby an isolated polynucleotide sequence, which hybridizes under stringentconditions to a polynucleotide sequence included in plasmid pALK3094comprising the nucleotide sequence SEQ ID No:17, deposited in E. coliRF8791 under accession number DSM 24410.

In the present invention the Malbranchea protease gene was isolated witha probe prepared by PCR using stringent hybridization as described inExample 1d. Standard molecular biology methods can be used in isolationof cDNA or a genomic DNA of the host organism, e.g. the methodsdescribed in the molecular biology handbooks, such as Sambrook andRussell, 2001.

Hybridization with a DNA probe, such as for example SEQ ID NO:11, SEQ IDNO:12 or SEQ ID NO:17 consisting of more than 100-200 nucleotides, isusually performed at “high stringency” conditions, i.e. hybridization ata temperature, which is 20-25° C. below the calculated meltingtemperature (Tm) of a perfect hybrid, the Tm calculated according toBolton and McCarthy (1962). Usually prehybridization and hybridizationare performed at least at 65° C. in 6×SSC (or 6×SSPE), 5×Denhardt'sreagent, 0.5% (w/v) SDS, 100 μg/ml denatured, fragmented salmon spermDNA. Addition of 50% formamide lowers the prehybridization andhybridization temperatures to 42° C. Washes are performed in low saltconcentration, e.g. in 2×SSC-0.5% SDS (w/v) for 15 minutes at roomtemperature (RT), followed in 2×SSC-0.1% SDS (w/v) at RT, and finally in0.1×SSC-0.1% SDS (w/v) at least at 65° C., or in conditions described inExample 1d.

According to one preferred embodiment the fungal serine protease enzymeof the invention is encoded by an isolated nucleic acid molecule, whichencodes a polypeptide comprising the amino acid sequence characterizedin SEQ ID No:18, or a polypeptide having at least 66% identity to theamino acid sequence SEQ ID No:18. Preferred enzymes show at least 66%,preferably at least 70%, more preferably at least 75%, even morepreferably at least 80% identity. Still more preferably the amino acidsequences show at least 85% or at least 90% or 95%, more preferably atleast 98%, most preferably 99% identity to the amino acid sequence ofSEQ ID No:18. The identities of the enzymes are compared using thecorresponding mature sequence regions.

Thus, within the scope of the invention is a polypeptide sequence, whichis encoded by a nucleic acid molecule encoding the amino acid sequenceof the full-length serine protease of the invention including theprepeptide (signal sequence) and the propeptide in addition to themature form of the enzyme, and which amino acid sequence ischaracterized in SEQ ID No:14.

Also, within the scope of the invention is a polypeptide sequence, whichis encoded by a nucleic acid molecule encoding the propeptide form ofserine protease enzyme of the invention including the propeptide inaddition to the mature form of the enzyme, and which amino acid sequenceis characterized in SEQ ID No:16.

One preferred embodiment of the invention is the fungal serine proteaseenzyme encoded by an isolated nucleic acid molecule, which comprises thenucleotide sequence encoding the mature form of the Malbranchea ALKO4122serine protease having SEQ ID No:18.

According to one preferred embodiment the fungal serine protease enzymeof the invention is encoded by an isolated nucleic acid moleculecomprising the nucleotide sequence SEQ ID No:17 encoding the mature formof the Malbranchea ALKO4122 enzyme (SEQ ID No:18).

Thus, within the scope of the invention is the polypeptide encoded bythe nucleic acid molecule having the nucleotide sequence SEQ ID No:13comprising the “coding sequence” for the enzyme. The expression “codingsequence” means the nucleotide sequence which initiates from thetranslation start codon (ATG) and stops at the translation stop codon(TAA, TAG or TGA). The translated full-length polypeptide starts usuallywith methionine and may comprise intron regions.

Also, within the scope of the invention is a fungal serine proteaseenzyme encoded by a nucleic acid molecule comprising the nucleotidesequence SEQ ID NO:15, which encodes the Malbranchea ALKO4122 proenzymeform.

According to another preferred embodiment of the invention the fungalserine protease is encoded by the polynucleotide sequence included inplasmid pALK3094 comprising the nucleotide sequence SEQ ID No:13 in E.coli RF8791, deposited under accession number DSM 24410.

One embodiment of the invention is the serine protease enzyme producedfrom a recombinant expression vector comprising the nucleic acidmolecule, which encodes the fungal serine protease enzyme ascharacterized above operably linked to regulatory sequences capable ofdirecting the expression of said serine protease enzyme in a suitablehost. Construction of said recombinant expression vector and use of saidvector is described in more detail in Example 2.

Suitable hosts for production of the fungal serine protease enzyme arehomologous or heterologous hosts, such as the microbial hosts includingbacteria, yeasts and fungi. Filamentous fungi, such as Trichoderma,Aspergillus, Fusarium, Humicola, Chrysosporium Neurospora, Rhizopus,Penicillium, Myceliophthora and Mortiriella, are preferred productionhosts due to the ease of down-stream processing and recovery of theenzyme product. Suitable hosts include species such as T. reesei, A.niger, A. oryzae, A. sojae, A. awamori or A. japonicus type of strains,F. venenatum or F. oxysporum, H. insolens or H. lanuginosa, N. crassaand C. lucknowense, some of which are listed as enzyme production hostorganisms in e.g. AMFEP 2009 list of commercial enzymes(http://www.amfep.org/list.html). More preferably, the enzyme isproduced in a filamentous fungal host of the genus Trichoderma orAspergillus, such as T. reesei or A. niger, A. oryzae or A. awamori.According the most preferred embodiment of the invention the fungalserine protease enzyme is produced in T. reesei.

The present invention relates also to an isolated nucleic acid moleculecomprising a polynucleotide sequence encoding the serine protease enzymeselected from the group consisting of:

-   -   (g) a nucleic acid molecule encoding a polypeptide having serine        protease activity and comprising the amino acid sequence as        depicted in SEQ ID No:18;    -   (h) a nucleic acid molecule encoding a polypeptide having serine        protease activity and at least 66% identity to the amino acid        sequence of SEQ ID No:18;    -   (i) a nucleic acid molecule comprising the coding sequence of        the nucleotide sequence as depicted in SEQ ID No: 17;    -   (j) a nucleic acid molecule comprising the coding sequence of        the polynucleotide sequence contained in DSM 24410;    -   (k) a nucleic acid molecule the coding sequence of which differs        from the coding sequence of a nucleic acid molecule of any one        of (c) to (d) due to the degeneracy of the genetic code; and    -   (l) a nucleic acid molecule hybridizing under stringent        conditions to a nucleic acid molecule contained in DSM 24426, or        SEQ ID No:17 encoding a polypeptide having serine protease        activity and an amino acid sequence which shows at least 66%        identity to the amino acid sequence as depicted in SEQ ID No:18.

The nucleic acid molecule of the invention may be RNA or DNA, whereinthe DNA may constitute of the genomic DNA or cDNA.

Standard molecular biology methods can be used in isolation and enzymetreatments of the polynucleotide sequence encoding the fungal serineprotease of the invention, including isolation of genomic and plasmidDNA, digestion of DNA to produce DNA fragments, sequencing, E. colitransformations etc. The basic methods are described in the standardmolecular biology handbooks, e.g. Sambrook and Russell, 2001.

Isolation of the Malbranchea protease gene encoding the MalbrancheaALKO4122 polypeptide is described in Example 1. Briefly, the PCRfragment obtained by using the degenerate oligonucleotide primers (SEQID No: 5 and SEQ ID No: 4) in the PCR reaction was used to isolate theprotease gene from Malbranchea ALKO4122. The genomic fragment includingthe protease gene was ligated into pBluescript II KS+ vector. Thefull-length Malbranchea protease gene was included in the plasmidpALK3094 deposited in E. coli to the DSMZ culture collection underaccession number DSM 24410. The deduced amino acid sequence of theserine protease was analyzed from the DNA sequence.

The nucleotide sequence of Malbranchea ALKO4122 protease (SEQ ID No: 13)its partial promoter and terminator sequences and the deduced amino acidsequence (SEQ ID No:14) are presented in FIG. 1A-B. The length of thegene is 1436 by (including the stop codon). Three putative introns werefound having the length of 72, 87 and 71 bps. The deduced proteinsequence consists of 401 amino acids including a predicted signalsequence of 20 amino acids (SignalP V3.0; Nielsen et al., 1997 andNielsen and Krogh, 1998) and a predicted propeptide from Gly21 toAsp120. The predicted molecular mass was 28.5 kDa for the maturepolypeptide and the predicted pI was 6.15. These predictions were madeusing the Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003).The deduced amino acid sequence contained three possible N-glycosylationsites (Asn134, Asn172 and Asn277), but according to CBS Server NetNGlycV1.0 only two sites, Asn134 and Asn277 are likely. The homologies to thepublished protease sequences were searched using the BLASTP program,version 2.2.25 at NCBI (National Center for Biotechnology Information)(Altschul et al., 1990). The identity values of the mature Malbrancheaprotease sequence to the corresponding regions of the most homologoussequences were obtained by using ClustalW2 alignment (Matrix: Gonnet,Gap open: 10, Gap extension: 0.20, Gap distances 5; available e.g. inwww.ebi.ac.uk/Tools/msa/clustalw2/). The results are shown in Table 2.

The highest identity values obtained from the BLASTP search for themature Malbranchea ALKO4122 protease of the present invention (SEQ IDNO: 18) were as follows: 66%, for Coccidioides posadasii putativesubtilisin-like protease (EER24932.1) and Coccidioides immitishypothetical protein CIMG^(—)09197 (XP_(—)001239485.1), 65% forUncinocarpus reesii hypothetical protein UREG_(—)05170 (EEP80328.1), 64%for Coccidioides immitis hypothetical protein CIMB_(—)01394(XP_(—)001247623.1), Coccidioides posadasii putative subtilisin-likeprotease (EER23662.1), Uncinocarpus reesii hypothetical protein(EEP81307.1) and Arthroderma otae alkaline proteinase (EEQ28657.1). Thehighest identities for the sequences in patent division were 55% for SEQID NO:2 in U.S. Pat. No. 5,962,765 (AAE30270.1; protease fromMetarhizium anisopliae) and SEQ ID:15 in WO 8807581 (AAA54276.1;protease from Tritirachium album). The mature Malbranchea ALKO4122protease sequence (SEQ ID NO:18) was aligned with the mature sequencesof the above homologous sequences using ClustalW2 alignment. Theidentity values (score %) obtained by using ClustalW2 alignment(http://vvww.ebi.ac.uk/Tools/msa/clustalw2/) obtained were from 63%-65%,

Thus, within the scope of the invention is an isolated polynucleotidesequence or isolated nucleic acid molecule, which encodes a fungalserine protease enzyme or polypeptide comprising the amino acid sequenceof the mature form of the Malbranchea ALKO4122 enzyme characterized inSEQ ID No: 18, 15, i.e. amino acids Ala121 to Arg401 of the full lengthserine protease of SEQ ID No:14.

The nucleic acid molecule is preferably a molecule comprising the codingsequence as depicted in SEQ ID No:17, which encodes the mature form ofthe fungal serine protease enzyme of this invention.

The isolated nucleic acid molecule of the invention may be a moleculecomprising the coding sequence of the polynucleotide sequence containedin DSM 24410, DSM 24426 or DSM 24427. DSM 24426 carries the nucleotidesequence of the PCR fragment (SEQ ID No:11) used in cloning the fulllength Malbranchea ALKO4122 protease gene. DSM 24427 carries thenucleotide sequence of the PCR fragment (SEQ ID No:12) obtained fromMalbranchea ALKO4178. DSM 24410 carries the nucleotide sequence of thefull length Malbranchea ALKO4122 protease gene (SEQ ID No:13).

The nucleic acid molecule of the invention may also be an analogue ofthe nucleotide sequence characterized in above. The “degeneracy” meansanalogues of the nucleotide sequence, which differ in one or morenucleotides or codons, but which encode the recombinant protease of theinvention.

The nucleic acid molecule may also be a nucleic acid moleculehybridizing under stringent conditions to a PCR probe contained inplasmids pALK3092 or pALK3093 deposited in E. coli under the accessionnumbers DSM 24426 and DSM 24427, respectively, or to a DNA sequence SEQID NO: 17 encoding a mature polypeptide having serine protease activityand an amino acid sequence. The hybridizing DNA may originate from afungus belonging to species Malbranchea or it may originate from otherfungal species.

Thus, within the scope of the invention is an isolated nucleic acidmolecule comprising a nucleotide sequence as depicted in SEQ ID No:17and analogues thereof.

The present invention relates also to a recombinant expression vector orrecombinant expression construct, which can be used to propagate orexpress the nucleic acid sequence encoding the chosen serine protease ina suitable prokaryotic or eukaryotic host. The recombinant expressionvector comprises DNA or nucleic acid sequences which facilitate ordirect expression and secretion of the serine protease encoding sequencein a suitable host, such as promoters, enhancers, terminators (includingtranscription and translation termination signals) and signal sequencesoperably linked the polynucleotide sequence encoding said serineprotease. The expression vector may further comprise marker genes forselection of the transformant strains or the selection marker may beintroduced to the host in another vector construct by co-transformation.Said regulatory sequences may be homologous or heterologous to theproduction organism or they may originate from the organism, from whichthe gene encoding the serine protease is isolated.

Examples of promoters for expressing the serine protease of theinvention in filamentous fungal hosts are the promoters of A. oryzaeTAKA amylase, alkaline protease ALP and triose phosphate isomerase,Rhizopus miehei lipase, Aspergillus niger or A. awamori glucoamylase(glaA), Fusarium oxysporum trypsin-like protease, Chrysosporiumlucknowense cellobiohydrolase 1 promoter, Trichoderma reeseicellobiohydrolase I (Cel7A) etc.

In yeast, for example promoters of S. cerevisiae enolase (ENO-1),galactokinase (GAL1), alcohol dehydrogenase (ADH2) and3-phosphoglycerate kinase can be used to provide expression.

Examples of promoter sequences for directing the transcription of theserine protease of the invention in a bacterial host are the promoter oflac operon of Escherichia coli, the Streptomyces coelicolor agarase dagApromoter, the promoter of the B. licheniformis alpha-amylase gene(amyL), the promoter of the B. stearothermophilus maltogenic amylasegene (amyM), the promoters of the B. sublitis xylA and xylB genes, etc.

Suitable terminators include those of the above mentioned genes or anyother characterized terminator sequences.

Suitable transformation or selection markers include those whichcomplement a defect in the host, for example the dal genes from B.subtilis or B. licheniformis or Aspergillus amdS and niaD. The selectionmay be based also on a marker conferring antibiotic resistance, such asampicillin, kanamycin, chloramphenicol, tetracycline, phleomycin orhygromycin resistance.

Extracellular expression of the serine protease of the invention ispreferable. Thus, the recombinant vector comprises sequencesfacilitating secretion in the selected host. The signal sequence of theserine protease of the invention or the presequence or prepeptide may beincluded in the recombinant expression vector or the natural signalsequence may be replaced with another signal sequence capable offacilitating expression in the selected host. Thus, the chosen signalsequence may be homologous or heterologous to the expression host. Alsoa natural propeptide may be replaced with another propeptide. Thepropeptide may be homologous or heterologous to the expression host.

Examples of suitable signal sequences are those of the fungal or yeastorganisms, e.g. signal sequences from well expressed genes. Such signalsequences are well known from the literature.

The recombinant vector may further comprise sequences facilitatingintegration of the vector into the host chromosomal DNA to obtain stableexpression and/or to facilitate targeting to a certain position in thehost genome.

The Malbranchea ALKO4122 protease of the invention with its own signalsequence was expressed from the T. reesei cbh1 (cel7A) promoter asdescribed in Example 2. The expression construction used to transformthe T. reesei host included also cbh1 terminator and synthetic amdSmarker for selecting the transformants from the untrasformed cells.

The present invention relates also to host cells comprising therecombinant expression vector as described above. Suitable hosts forproduction of the fungal serine protease enzyme are homologous orheterologous hosts, such as the microbial hosts including bacteria,yeasts and fungi. Production systems in plant or mammalian cells arealso possible.

Filamentous fungi, such Trichoderma, Aspergillus, Fusarium, Humicola,Chrysosporium, Neurospora, Rhizopus, Penicillium, Myceliophthora andMortiriella, are preferred production hosts due to the ease ofdown-stream processing and recovery of the enzyme product. Suitableexpression and production host systems are for example the productionsystem developed for the filamentous fungus host Trichoderma reesei (EP244234), or Aspergillus production systems, such as A. oryzae or A.niger (WO 9708325, U.S. Pat. No. 5,843,745, U.S. Pat. No. 5,770,418), A.awamori, A. sojae and A. japonicus-type strains, or the productionsystem developed for Fusarium, such as F. oxysporum (Malardier et al.,1989) or F. venenatum, and for Neurospora crassa, Rhizopus miehei,Mortiriella alpinis, H. lanuginosa or H. insolens or for Chrysosporiumlucknowense (U.S. Pat. No. 6,573,086). Suitable production systemsdeveloped for yeasts are systems developed for Saccharomyces,Schizosaccharomyces or Pichia pastoris. Suitable production systemsdeveloped for bacteria are a production system developed for Bacillus,for example for B. subtilis, B. licheniformis, B. amyloliquefaciens, forE. coli, or for the actinomycete Streptomyces. Preferably the serineprotease of the invention is produced in a filamentous fungal host ofthe genus Trichoderma or Aspergillus, such as T. reesei, or A. niger, A.oryzae, A. sojae, A. awamori or A. japonicus-type strains. According themost preferred embodiment of the invention the fungal serine proteaseenzyme is produced in T. reesei.

The present invention relates also to a process for producing apolypeptide having serine protease activity, said process comprising thesteps of culturing the natural or recombinant host cell carrying therecombinant expression vector for a serine protease of the inventionunder suitable conditions and optionally isolating said enzyme. Theproduction medium may be a medium suitable for growing the host organismand containing inducers for efficient expression. Suitable media arewell-known from the literature.

The invention relates to a polypeptide having serine protease activity,said polypeptide being encoded by the nucleic acid molecule of theinvention and which is obtainable by the process described above.

The invention further relates to a process for obtaining an enzymepreparation comprising a polypeptide, which has serine proteaseactivity, said process comprising the steps of culturing a host cellcarrying the expression vector of the invention and either recoveringthe polypeptide from the cells or separating the cells from the culturemedium and obtaining the supernatant having serine protease activity.

The present invention relates also to an enzyme preparation, whichcomprises the serine protease enzyme characterized above. The enzymepreparation or composition has serine protease activity and isobtainable by the process according to the invention.

Within the invention is an enzyme preparation as well as compositioncomprising the serine protease of the invention.

The enzyme preparation or composition (e.g. detergent formulation)containing the protease enzyme of the invention may further compriseother enzymes selected from the group consisting of proteases (otherprotease than that of the invention), amylases, lipases, cellulases,cutinases, pectinases, mannanases, xylanases and oxidases, such as alaccase or peroxidase with or without a mediator. These enzymes areexpected to enhance the performance of the serine proteases of theinvention e.g. by removing the carbohydrates and oils or fats present inthe material to be handled. Said enzymes may be natural or recombinantenzymes produced by the host strain or may be added to the culturesupernatant after the production process.

Said enzyme preparation or composition may further comprise one or moresuitable additives selected from the group consisting of surfactants orsurface active agents, buffers, anti-corrosion agents, stabilizers,bleaching agents, mediators, builders, caustics, abrasives andpreservatives, optical brighteners, antiredeposition agents, dyes,pigments, perfumes etc.

Surfactants are useful in emulsifying grease and wetting surfaces. Thesurfactant may be a non-ionic including semi-polar and/or anionic and/orcationic and/or zwitterionic.

Buffers may be added to the enzyme preparation or composition to modifypH or affect performance or stability of other ingredients.

Suitable stabilizers include polyols such as propylene glycol orglycerol, a sugar or sugar alcohol, lactic acid, boric acid, or boricacid derivatives, peptides, etc.

Bleaching agent is used to oxidize and degrade organic compounds.Examples of suitable chemical bleaching systems are H₂O₂ sources, suchas perborate or percarbonate with or without peracid-forming bleachactivators such as tetraacetylethylenediamine, or alternativelyperoxyacids, e.g. amide, imide or sulfone type. Chemical oxidizers maybe replaced partially or completely by using oxidizing enzymes, such aslaccases or peroxidases. Many laccases do not function effectively inthe absence of mediators.

Builders or complexing agents include substances, such as zeolite,diphosphate, triphosphate, carbonate, citrate, etc. The enzymepreparation may further comprise one or more polymers, such ascarboxymethylcellulose, poly(ethylene glycol), poly(vinyl alcohol),poly(vinylpyrrolidone), etc. Also, softeners, caustics, preservativesfor preventing spoilage of other ingredients, abrasives and substancesmodifying the foaming and viscosity properties can be added.

According to one preferred embodiment of the invention said enzymepreparation or composition comprising said enzyme is in the form ofliquid, powder, granulate or tablets. According to a preferredembodiment of the invention said composition is a detergent formulationin the form of liquid, powder, granulate or tablets. Further, the enzymein the preparation or composition may be in the form of immobilizedenzyme.

The serine protease of the present invention may like other proteases,particularly alkaline proteases be used in the detergent, protein,brewing, meat, photographic, leather, dairy and pharmaceuticalindustries (Kalisz, 1988; Rao et al., 1998). For example, it may be usedas an alternative to chemicals to convert fibrous protein waste (e.g.horn, feather, nails and hair) to useful biomass, protein concentrate oramino acids (Anwar and Saleemuddin, 1998). The use of the serineprotease of the present invention may like other enzymes provesuccessful in improving leather quality and in reducing environmentalpollution and saving energy and it may like alkaline proteases be usefulin synthesis of peptides and resolution of the mixture of D,L-aminoacids. Subtilisin in combination with broad-spectrum antibiotics in thetreatment of burns and wounds is an example of the use of serineproteases in pharmaceutical industry, therefore the fungal serineprotease of the present invention may also find such use and may alsolike alkaline proteases be applicable in removal of blood on surgicalequipments and cleaning contact lenses or dentures. Like alkalineprotease from Conidiobolus coronatus, the fungal serine protease of thepresent invention may be used for replacing trypsin in animal cellcultures. The proteases of the invention can also be used in cleaning ofmembranes and destruction of biofilms. In baking the proteases can beused e.g. in destruction of the gluten network and in other foodapplications in hydrolysis of food proteins, e.g proteins in milk. Theycan also be used e.g. in treating yeast, rendering (extracting moreprotein from animal bones), creating new flavours, reducing bitterness,changing emulsifying properties, generating bioactive peptides andreducing allergenicity of proteins. The substrates include animal, plantand microbial proteins.

Detergent industry, particularly the laundry detergent industry, hasemerged as the single major consumer of proteases active at high pHrange (Anwar and Saleemuddin, 1998). The ideal detergent protease shouldpossess broad substrate specificity to facilitate the removal of largevariety of stains due to food, grass, blood and other body secretions.It has to be active in the pH and ionic strength of the detergentsolution, the washing temperature and pH, and tolerate mechanicalhandling as well as the chelating and oxidizing agents added todetergents. Due to awareness for energy conservation, it is currentlydesirable to use the protease at lower temperatures.

The present invention relates also to the use of the serine proteaseenzyme or the enzyme preparation for detergents, treating textilefibers, for treating proteinaceous materials, such as wool, hair, silk,leather, for treating feed or food, or for any application involvingmodification, degradation or removal of proteinaceous material.

One preferred embodiment of the invention is therefore the use of theserine protease enzyme as characterized above as a detergent additiveuseful for laundry detergent and dish wash compositions, includingautomatic dish washing compositions.

The expression “detergent” is used to mean substance or materialintended to assist cleaning or having cleaning properties. The term“detergency” indicates presence or degree of cleaning property. Thedegree of cleaning property can be tested on different proteinaceous orprotein containing substrate materials or stains or stain mixtures boundto solid, water-insoluble carrier, such as textile fibers or glass.Typical proteinaceous material includes blood, milk, ink, egg, grass andsauces. For testing purposes variety of proteinaceous stains arecommercially available. The function of the detergent enzyme is todegrade and remove the protein-containing stains. Test results depend onthe type of stain, the composition of the detergent and the nature andstatus of textiles used in the washing test (Maurer, 2004).

The term “low temperature” in context of the present application meanstemperature ranges from 10° C. to 30° C., which according to theExperiments are not optimal for the performance of many of the presentlyavailable enzyme preparations, particularly the detergent enzymepreparations. By the term “moderate temperature” is meant a temperaturerange from 30° C. to 60° C.

The term “applicable at low or moderate temperature ranges” includesindustrial applications in which it is desirable that the enzymefunctions effectively at low or moderate temperature ranges (10° C. to60° C.). Such applications include their use in food, feed and leatherindustry, pharmaceuticals, diagnostics, waste management and silverrecovery. As meant herein, these applications exclude the use of theserine protease enzyme of the invention as a biocontrol agent inbiological control of plant pathogenic fungi and nematodes.

In the present invention the term “detergent stability” means that theenzyme or enzyme variant sufficiently retains its activity in detergentsolution, during storage and/or washing. Therefore it is efficient indegrading or removing proteinaceous stains or material in the presenceof a detergent such as the Ecolabel Reference Detergent, light duty (wfkTestgewebe GmbH) or the Commercial liquid detergent as described inTable 3 of Example 6. The stability may be assayed by determining theresidual activity e.g. after several days incubation (at 37° C.) indetergent. The residual protease activity may be determined using themethod described in Example 3 or any other method disclosed in theliterature (Gupta et al. 2002).

The term “effective amount” of a serine protease refers to the quantityof the protease enzyme necessary to achieve the enzymatic activity inthe specific detergent composition. Preferably the detergent compositionof the invention comprises from about 0.0001% to about 10% by weight ofthe detergent composition of a protease variant of the invention, morepreferably from 0.001% to about 1%, still more preferably from 0.001% toabout 0.5%.

Typically, the wash performance of protease is measured as “stainremoval efficiency” or “stain removal effect” or “degree of cleaningproperty” meaning a visible and measureable increase of lightness orchange in colour of the stained material, e.g. in artificially soiledswatches or test cloths. Lightness or change in colour values can bemeasured, for example by measuring the colour as reflectance values witha spectrophotometer using L*a*b* colour space coordinates as describedin Examples 6 to 8. Fading or removal of proteinaceous stain indicatingof the protease performance (stain removal efficiency) is calculated forexample as ΔL*, which means lightness value L* of enzyme treated fabricminus lightness value L* of fabric treated with washing liquor withoutenzyme (enzyme blank or control). The presence of detergent may improvethe performance of the enzyme in removing the stains. The serineprotease of the present invention degrades various kinds ofproteinaceous stains under alkaline conditions in the presence ofdetergents with different compositions (Examples 6-8).

As shown in Example 6 the serine protease of the invention removed theblood/milk/ink stain at 10-50° C. and especially at or below 30° C. inCommercial liquid detergent considerably better than the commercialprotease preparations Savinase® 16 L and Savinase® Ultra 16 L (FIGS. 6and 7). The enzyme preparations were dosed as activity units. The sameeffect was observed also when the dosing was calculated as amount ofadded protein (FIG. 8). It is surprising that Malbranchea ALKO4122protease shows optimal stain removal performance at very broadtemperature range and especially at low temperatures, like 10-30° C.,despite of its high temperature optimum at analytical conditions oncasein substrate (approx. 70° C., FIG. 5A). Savinase® that has similartemperature profile in analytical conditions (FIG. 5A) shows clearlylower performance at cold washing temperatures. The results of thesetests indicate that the protease according to the invention hasexcellent performance with liquid detergent at broad temperature rangeand even at very cold washing temperatures.

In addition to the different blood/milk/ink stains the MalbrancheaALKO4122 protease was effective in removing stains, such as grass andcocoa when tested in liquid detergent at 30 and 60° C. Treatments wereperformed in ATLAS LP-2 Launder-Ometer and enzyme dosed as activity.Results (FIGS. 9A-H) show that the Malbranchea proteasea was effectiveon several stains at both temperatures 30 and 60° C. and showed betterstain removal performance compared to Savinase® Ultra 16 L. MalbrancheaALKO4122 was effective also on groundnut oil/milk and egg yolk stains.

The performance of the Malbranchea ALKO4122 protease was tested alsowith Commercial traditional detergent powder containing bleaching agentsand optical brighteners and with ECE Reference detergent 77 withoutoptical brighteners and bleaching agents at 50° C. at pH 10.5 or 10,respectively, as described in Example 8. The ability of the enzyme inremoving blood/mills/ink stain on polyester-cotton material was assayed.As shown in FIGS. 10 A and B the protease of the invention is suitablealso for powder detergents at very alkaline conditions and the effectwas similar compared to Savinase® Ultra 16 L, when each enzymepreparation was dosed as activity units.

In addition to washing, the enzyme of the present invention sufficientlyretains its activity also during storage, even when stored in liquiddetergents, as shown in Examples 9 and 10. Malbranchea ALKO4122 proteasehas excellent storage stability at 37° C. compared to e.g. Fusariumprotease Fe_RF6318 (WO2010125174A1) (FIGS. 11A and B). FIGS. 12A and 12Bshow that Malbranchea protease has especially good stability in EcolabelReference Detergent, light duty (wfk Testgewebe GmbH) and Commercialliquid detergent (Table 3) compared to Fusarium protease and similarstability in Ecolabel compared to commercial bacterial proteaseSavinase® 16 L. Good stability of the protease according to theinvention at elevated temperatures makes it especially suitable forliquid detergent formulations in warm climate countries.

According to a preferred embodiment of the invention the fungal serineprotease of the invention is useful in detergent liquids and detergentpowders as shown in Examples 6 to 10. The enzyme of enzyme preparationof the invention may be formulated for use in a hand or machine laundryor may be formulated for use in household hard surface cleaning orpreferably in hand or machine dishwashing operations.

As a conclusion it can be seen that a new thermostable fungal serineprotease enzyme is provided, originating from a thermophilicmicro-organism, and which acts particularly well at low temperatures andis compatible and stable in liquid detergent compositions, whereby lessstabilizing and other additives are needed.

The invention is illustrated with the following examples relating tosome embodiments of the invention, however, the invention is not meantto be limited to these examples only.

EXAMPLES Example 1 Synthesis of Probes for Cloning Protease Genes fromMalbranchea and Cloning of Protease Gene from Malbranchea ALKO4122 andALKO4178 Strains (a) Isolation of DNA and Molecular Biology Methods Used

Standard molecular biology methods were used in the isolation and enzymetreatments of DNA (e.g. isolation of plasmid DNA, digestion of DNA toproduce DNA fragments), in E. coli transformations, sequencing etc. Thebasic methods used were either as described by the enzyme, reagent orkit manufacturer or as described in the standard molecular biologyhandbooks, e.g. Sambrook and Russell (2001). Isolation of genomic DNAfrom filamentous fungi was done as described in detail by Raeder andBroda (1985).

(b) Primers for Probe Preparation

The probes for cloning the genes encoding Malbranchea proteases weresynthesised by PCR. Degenerate sense and antisense oligos were plannedbasing on the consensus amino acid sequences of fungal proteases (e.g.Fusarium equiseti RF6318, Fusarium acuminatum RF7182, T. reesei Prbl inWO2010125174, WO2010125175 and WO2011003968, AB Enzymes Oy,respectively). An additional 5′-primer (DET5) was synthesised accordingto a region chosen from the published N-terminal sequence of Malbrancheacinnamomea Thermomycolin (altogether 34 amino acids; Swiss-Protaccession number P13858.1). Amino acids 20-28 of the above sequence wereused for designing DET5. The sequences of the primers and the peptidesequences used for their design are shown in the following Table 1 (SEQID NOs: 1-5 and SEQ ID NOs: 6-10, respectively). The oligo's name, SEQID NO, length, degeneracy and nucleotide sequence are included in thetable as well as the peptide sequences used in planning of the primersand their SEQ ID NOs: 6-10.

TABLE 1 Oligonucleotides (SEQ ID NOs: 1-5) used as PCR primers in probeamplification. SEQ SEQ ID Length ID Oligo NO: (nts) DegeneracySequence^((a) Peptide NO: DET1 1 17  256 GGNCAYGGNACNCAYGT (s) GHGTHV  6DET2 2 20  128 AAYTGGGCNGTNAAYGAYAT (s) NWAVNDI  7 DET3 3 20 3072NCCRTARTTNGTRAANSWNG (as) ASFTNYG  8 DET4 4 20 6912NGCCATNSWNGTNCCNSWDA (as) ISGTSMA  9 DET5 5 26 6144CARGCNGGNATHMGNGAYTAYCA QAGIRDYHY 10 YTA (s) ^((a)N = A, T, C or G; R =A or G, Y = T or C, H = A, C or T, M = A or C; “s” and “as” inparenthesis after the nucleotide sequence = sense strand and antisensestrand, respectively.

(c) PCR Reactions and Selection of Probes for Cloning the Full-lengthProtease Genes

Genomic DNA preparations isolated from Malbranchea ALKO4122 and ALKO4178were used as templates in the PCR reactions. Malbranchea ALKO4122 hasbeen identified by CBS identification service (Utrecht, The Netherlands)as Malbranchea cinnamomea (Lib.) Oorschot de Hoog (Synonym ofMalbranchea pulchella var. sulfurea (Miehe) Cooney & R. Emers.) atJanuary, 2011. Malbranchea ALKO4178 is deposited to Roal Oy culturecollection as Malbranchea sulfurea but no further identification of thisisolate has been done by CBS or other institute specialised inidentification of filamentous fungi.

For isolation of the genomic DNAs, Malbranchea ALKO4122 and ALKO4178mycelia were grown by cultivating the strains at 37° C. for 4 days at250 rpm in 50 ml of glucose-yeast extract medium (25 g glucose, 27.5 gyeast extract pH 6.5). The mycelia were collected and the genomic DNAswere isolated and used as templates for probe synthesis. The PCRreaction mixtures contained Phusion® GC buffer (Finnzymes/FisherScientific, Finland), 0.2 mM dNTPs, 50 μmol each primer, DMSO (1/10 ofthe volume) and 1 units of Phusion® polymerase (Finnzymes/FisherScientific) and approximately 0.5-1 μg of genomic DNA per 50 μl reactionvolume. The conditions for the PCR reactions were: 1 min initialdenaturation at 98° C., followed by 28 cycles of 10 sec at 98° C., 30sec annealing at 45, 47, 52 and 57° C., 30 sec extension at 72° C. and afinal extension at 72° C. for 5 min. Primer combination DET5 (SEQ ID NO:5) and DET4 (SEQ ID NO: 4) produced, in the three lowest annealingtemperatures (see above), a DNA product having size of about 0.8 kb.This size corresponded to the size expected to be obtained from aprotease gene, according to calculations basing on published fungalprotease sequences. A product with similar size was obtained when eitherALKO4122 or ALKO4178 genomic DNA was used as a template. The DNAproducts were isolated and purified from the PCR reaction mixtures andligated to pCR®4 Blunt-TOPO vector according to the manufacturer'sinstructions (Invitrogen, USA). The resulting plasmids were named aspALK3092 (ALKO4122 PCR product as an insert) and pALK3093 (ALKO4178 PCRproduct as an insert). The inserts were sequenced from both pALK3092(SEQ ID NO: 11) and pALK3093 (SEQ ID NO: 12). Both the inserts werefound to be 791 by in length and they only showed one nucleotidedifference with each other in the synthesized region (disregarding theprimer sequences). The nucleotide 323 in the pALK3092 insert was C andin the pALK3093 insert it was T. The E. coli strains RF8758 and RF8759including the plasmids pALK3092 and pALK3093, respectively, weredeposited to the DSMZ collection under the accession numbers DSM 24426and DSM 24427, respectively.

A BLASTP search was done with the sequences obtained using programversion 2.2.23 at NCBI (National Center for Biotechnology Information)with default settings (Altschul et al., 1990). The encoded amino acidsequences showed homology e.g. to hypothetical and conservedhypothetical proteins from Uncinocarpus reesii (XP_(—)002584481.1 andXP_(—)002583205.1, respectively) and to a putative subtilisin-likeprotein from Coccidioides posadasii (EER24932.1) and hypotheticalprotein from Coccidioides immitis (XP_(—)001239485.1) with identities of59-65%. Thus, the results indicate that the DNA fragments obtained fromthe PCR reactions were parts of genes encoding proteases and thus usefulas probes for screening the full-length protease gene(s) fromMalbranchea. The encoded amino acid sequence that was following thesequence encoded by the primer DET5 was, however, not identical with thepublished Malbranchea Thermomycolin sequence (amino acids 29-34,accession number P13858).

(d) Cloning of the Full-length Malbranchea ALKO4122 Protease Gene

Malbranchea ALKO4122 and ALKO4178 genomic DNAs were digested withseveral restriction enzymes for Southern blot analysis. The digestionswere run on gel and Southern transfer and hybridization were performed.The probe for Southern blots was labelled by PCR using M13 reverse andM13 forward primers and pALK3092 (Example 1c) as a template. The probesequence includes the SEQ ID NO:11 (Example 1c). The labelling of theprobe using PCR DIG (digoxigenin) labeling mix and hybridization wereperformed according to supplier's instructions (Roche, Germany).Hybridization was performed over night at 68° C. After hybridization thefilters were washed as follows: 2×5 min washes at room temperature using2×SSC-0.1 SDS, followed by 2×15 min washes at 68° C. using 0.1×SSC-0.1%SDS.

Several hybridizing fragments in the Malbranchea ALKO4122 and ALKO4178genomic DNA digests were detected. Most of the digests gave identicalresults from both the genomes. A hybridising approximately 2.7 kbfragment was obtained from the genomic XbaI digest. The XbaI fragmentwas analysed to contain the full-length protease gene (by doubledigesting the genomic DNA with XbaI and PstI included in the probesequence and by size calculations basing on the published fungalprotease sequences). The DNA fragments were isolated from ALKO4122genomic XbaI digestion from the size range of the hybridizing fragment(approximately 2.7 kb). Isolation was done from an agarose gel. Theisolated genomic fragments were cloned to pBluescript II KS+(Stratagene, USA) vector cleaved with XbaI. Ligation mixture wastransformed into Escherichia coli XL10-Gold cells (Stratagene) andplated on LB (Luria-Bertani) plates containing 50 μg/ml ampicillin. Thepositive E. coli colonies were screened using colonial hybridizationwith PCR labeled pALK3092 insert as a probe. Hybridization was performedas described for the genomic DNA digests. Altogether 20 clones werepicked from the plates and out of these eight were shown, by XbaIrestriction digestion and Southern blot hybridization (done as describedfor the genomic hybridization), to contain inserts having the expectedsize and hybridizing with the pALK3092 probe. The XbaI insert (2961 bp)was sequenced and confirmed to contain a full-length MalbrancheaALKO4122 protease gene (SEQ ID NO:13). The plasmid containing the aboveXbaI genomic fragment was named as pALK3094. The E. coli strain RF8791including the plasmid pALK3094 was deposited to the DSMZ collectionunder the accession number DSM 24410.

(e) Characterisation of the Gene Encoding Malbranchea ALKO4122 Proteaseand the Deduced Amino Acid Sequence of the Protease

The gene sequence (SEQ ID NO:13), its partial promoter and terminatorsequences and the deduced amino acid sequence of the encoded protease(SEQ ID NO:14) are shown in FIG. 1. The length of the gene is 1436 by(including the stop codon). Three putative introns were found havinglengths of 72, 87 and 71 bps. The introns had the consensus 5′ and 3′border sequences as well as the internal consensus sequences accordingto those identified from several fungal introns (Gurr et al., 1987). Thededuced protein sequence (SEQ ID NO:14) consists of 401 amino acids,including a predicted signal sequence of 20 amino acids (SignalP V3.0;Nielsen et al., 1997 and Nielsen and Krogh, 1998) and a predicted prosequence of 100 amino acids. The location of the N-terminus of themature protease, Ala 121, was estimated according to comparisons madewith other fungal protease sequences. The predicted molecular mass ofthe mature protease was 28 530 Da and the predicted pI was 6.15. Thesepredictions were calculated using the Compute pI/MW tool at ExPASyserver (Gasteiger et al., 2003). The deduced mature amino acid sequencecontained three possible N-glycosylation sites at amino acid positionsAsn134, Asn172 and Asn277 of the mature sequence (amino acid positions254, 292 and 397 in FIG. 1), but according to CBS Server NetNGlyc V1.0only the sites at positions Asn 134 and Asn277 are likely.

The N-terminal sequence of the Malbranchea ALKO4122 proteasecorresponded only partly to the previously published M. cinnamoneaThermomycolin N-terminal sequence (P13858): only the first 28 aminoacids were identical in these two sequences.

There was one nucleotide difference in the Malbranchea ALKO4122 proteasegene probe sequence in pALK3092 compared to the genomic ALKO4122protease gene sequence (in the corresponding region). The genomicALKO4122 protease gene sequence was, however, identical to that ofALKO4178 protease probe sequence in pALK3093 (in the correspondingregion). Thus, the one nucleotide difference in pALK3092 insert (Example1c) most probably resulted from a mutation in the probe PCR reaction.

(f) Cloning of the Malbranchea ALKO4178 Protease Gene

Primers DET27 (SEQ ID NO: 19) and DET28 (SEQ ID NO: 20) were designed tosynthesise the full-length protease gene from Malbranchea ALKO4178 byPCR. DET 27 is a sense primer from the promoter (from nucleotide −44 to−24 from ATG) and DET28 is an antisense primer from the terminator (fromnucleotide 55 to 36 from the stop codon) of the Malbranchea ALKO4122protease gene (FIG. 1). The PCR reaction mixtures contained 1× Phusion®HF buffer (Finnzymes/Fisher Scientific, Finland), 0.2 mM dNTPs, 75 μmoleach primer and 2 units of Phusion® polymerase (Finnzymes/FisherScientific) and approximately 1.5 μg of genomic DNA per 200 μl reactionvolume. The conditions for the PCR reactions were: 30 sec initialdenaturation at 98° C., followed by 24 cycles of 10 sec at 98° C., 30sec annealing at 60 and 65° C., 60 sec extension at 72° C. and a finalextension at 72° C. for 7 min. Fragments of expected length (˜1.5 kb)were obtained from the reactions. Fragments from two separate PCRreactions were isolated and ligated into pCR4®Blunt-TOPO® vectors. Theinserts were sequenced from the separate clones. Sequences of theinserts in both clones were identical with each other and with theMalbranchea ALKO4122 protease gene, its partial promoter and terminator(FIG. 1). The plasmid containing the Malbranchea ALKO4178 protease gene(the PCR fragment obtained) was named as pALK3147. The E. coli cloneincluding this plasmid was stored to Roal Oy culture collection asRF9332.

(g) Homology, Identity and Alignment Studies

The mature Malbranchea ALKO4122 protease amino acid sequence (SEQ IDNO:18) was used to search homologous protease sequences from publicsources. Both the redundant protein sequences and protein sequences inthe Patent division of GenBank were searched. The BLASTP program version2.2.25 at NCBI (National Center for Biotechnology Information) withdefault settings was used in the search (Altschul et al., 1990). Thehighest identities obtained from all redundant sequences were 66% forCoccidioides posadasii putative subtilisin-like protease (EER24932.1)and Coccidioides immitis hypothetical protein CIMG^(—)09197(XP_(—)001239485.1), 65% for Uncinocarpus reesii hypothetical proteinUREG_(—)05170 (EEP80328.1), 64% for Coccidioides immitis hypotheticalprotein CIMB_(—)01394 (XP_(—)001247623.1), Coccidioides posadasiiputative subtilisin-like protease (EER23662.1), Uncinocarpus reesiihypothetical protein (EEP81307.1) and Arthroderma otae alkalineproteinase (EEQ28657.1). The EER24932.1 and XP_(—)001239485.1 onlydiffer from each other by three amino acids and XP_(—)001247623.1 andEER23662.1 from each other only by one amino acid. The MalbrancheaALKO4122 protease sequence was aligned with the above homologoussequences using ClustalW2 alignment. The putative mature sequences fromeach protease were used in the alignment. The mature sequences from eachprotease were compared with each other. The identity values (score %)obtained by using ClustalW2 alignment(http://www.ebi.ac.uk/Tools/msa/clustalw2/) are shown in Table 2. Themature amino acid sequences excluding the signal peptides andpropeptides were aligned using default settings (Protein Weight Matrix:Gonnet, Gap open: 10, Gap extension: 0.20, Gap distances 5). SEQ IDNO:18 is the mature amino acid sequence of ALKO4122 protease. Theidentity values (score %) obtained were from 63%-65%.

The highest identities for the sequences in patent division were 55% forSEQ ID NO:2 in U.S. Pat. No. 5,962,765 (AAE30270.1; protease fromMetarhizium anisopliae) and SEQ ID:15 in WO 8807581 (AAA54276.1;protease from Tritirachium album).

TABLE 2 The identity values (score %) obtained from ClustalW 2.1multiple sequence alignment of the deduced protease amino acidsequences. Sequence 1 2 3 4 5 6 7 8 1 = SEQ ID NO: 18 100 2 = EER24932.165 100 3 = XP_001239485.1 65 99 100 4 = EEP80328.1 64 73 72 100 5 =XP_001247623.1 63 59 59 60 100 6 = EER23662.1 63 59 59 61 99 100 7 =EEP81307.1 63 59 59 63 62 61 100 8 = EEQ28657.1 64 58 58 58 57 57 62 100

Example 2 Production of the Recombinant Malbranchea ALKO4122 Protease inTrichoderma reesei (a) Preparing the Production Vector and ProductionStrains

The expression plasmid pALK3097 was constructed for production ofrecombinant Malbranchea ALKO4122 protease in Trichoderma reesei. Thegene with its own signal sequence was exactly fused to the T reesei cbh1(cel7A) promoter by PCR. The primers used in the PCR reaction were namedas DET17 (5′-primer; SEQ ID NO:21) and DET18 (3′-primer; SEQ ID NO:22).The DET17 primer contains a partial cbh1 promoter from SacII site(position −16 from ATG) to position −1 and the beginning of theMalbranchea protease gene (26 nucleotides including the ATG start codon)and 3 extra nucleotides at the 5′ end. The DET18 contains 26 nucleotidesfrom the end of the Malbranchea protease gene (including the STOP codon)and a linker including a BamHI site for fusing the gene from its 3′-endto pALK2777 linker (cbh1 terminator; see below). The protease gene'snative terminator is not included in the construction.

The protease gene was excised from the PCR fragment by SacII-BamHIdigestion and fused to pALK2777 expression vector backbone cleaved withthe same enzymes (FIG. 2). The pALK2777 plasmid contains the cbh1promoter (to SacII site), linker including e.g. the BamHI site, cbh1terminator and a synthetic amdS gene for screening the transformants.The synthetic amdS gene in pALK2777 contains a shortened terminator (toXbaI site) compared to the native amdS gene (Kelly and Hynes, 1985).Also, the introns of the native amdS gene have been removed and chosenrestriction sites from the amdS promoter and gene have been modified toease the construction and isolation of the expression cassettes.However, the amino acid sequence encoded by the synthetic amdS gene isidentical to that encoded by the wild type amdS gene.

The 7.2 kb linear expression cassette (presented in FIG. 3) was isolatedfrom the vector backbone after NotI digestion and was used fortransforming Trichoderma reesei protoplasts. The T. reesei host strainused does not produce any of the four major T. reesei cellulases (CBHI,CBHII, EGI, EGII). The transformations were performed as in Penttiläetal. (1987) with the modifications described in Karhunen et al. (1993).The transformants were purified on selection plates through singleconidia prior to sporulating them on PD.

(b) Protease Production in Shake Flasks and Laboratory Scale Bioreator

The transformants were inoculated from the PD slants to shake flaskscontaining 50 ml of complex lactose-based cellulase inducing medium(Joutsjoki et al., 1993) buffered with 5% KH₂PO₄ and pH 6.0. Theprotease production of the transformants was analyzed from the culturesupernatants after growing the transformants for 5 days at 30° C., 250rpm. In SDS-PAGE gels, a major protein band of about 30 kDacorresponding to the expected mass of recombinant protease was detectedfrom the spent culture supernatants. The protease activity was assayedfrom the samples using casein as a substrate as described in Example 3.Clearly increased activities compared to host were measured from theculture supernatants. The integration of the expression cassette intothe fungal genomes was confirmed from chosen transformants by usingSouthern blot analysis in which several genomic digests were used andthe expression cassette pALK3097 was used as a probe.

The T. reesei transformants producing the best protease activities inthe shake flask cultivations were chosen to be cultivated in laboratoryscale bioreactors. Cellulase inducing complex medium was used in thecultivations. The spent culture medium obtained from the cultivations orconcentrated samples were used as starting materials for purificationand further biochemical characterization of the recombinant MalbrancheaALKO4122 protease (Examples 3-5) as well as for the application testspresented in Examples 6-10.

Example 3 Protease Activity Assay

Protease activity was measured using casein as substrate. Rate of caseindegradation by a protease was measured by monitoring the release ofacid-soluble peptide fragments as a function of time. Acid-solublepeptides were quantified spectrophotometrically. The result wasexpressed as 1 μg of tyrosine per min per ml (or g).

First all reagent solutions needed in the assay were prepared indeionized water, Milli-Q or equivalent as follows.

(STW) Synthetic Tap Water:

The following stock solutions were prepared:

-   -   (A) 5.8 g CaCl₂×2 H₂O/200 ml H₂O    -   (B) 2.8 g MgCl₂×6 H₂O/200 ml H₂O    -   (C) 4.2 g NaHCO₃/200 ml H₂O

10 ml of these solutions were added in the given order to 300 ml of H₂Owith stirring, then made up to 1 liter with H₂O. The resulting solutionwas called as synthetic tap water.

Tris Solution, 0.3 M in Synthetic Tap Water:

36.3 g of Trizma base (SIGMA T-1503) was dissolved in synthetic tapwater and made up to 1 liter.

Casein Solution:

6 g of casein (Hammarsten Usb. 12840) was added to 350 ml synthetic tapwater and dissolved with magnetic stirring for 10 min. 50 ml of Trissolution was added and the solution was stirred for another 10 min.Then, the solution was heated up to 70° C. After that the temperaturewas let to decrease to 50° C. and the pH was adjusted to 8.5 with 0.1MNaOH. Stirring was continued until room temperature was reached. Thesolution was made up to 500 ml with synthetic tap water. The substratesolution was stored for maximum of 3 days in refrigerator (or stored asfrozen).

110 mM Trichloroacetic Acid Reagent (Reaction Stop Solution):

18 g of TCA (Merck 807) was dissolved in H₂O and made up to 1 liter.

0.5 M Na₂CO:

53 g of Na₂CO₃ was dissolved in H₂O and made up to 1 liter.

Folin Solution:

25 ml of 2N Folin-Ciocalteu's phenol reagent (SIGMA, F 9252) was dilutedup to 100 ml with H₂O.

Sample Dilution Buffer:

The sample was diluted in 50 mM Tris-HCl buffer pH 8.5.

The most suitable dilution will yield an absorbance of 0.4-0.8 in thereaction.

Assay:

The assay was started by temperating 2.5 ml of substrate solution intest tubes for 5 min at 50° C. After that 0.5 ml of diluted enzymesolution was added, mixed with vortex mixer and the reaction wasconducted at 50° C. for exactly 30 min. The enzyme blank was preparedlike the sample but the reaction stop solution (110 mM TCA) was added intest tube before the sample. After the reaction 2.5 ml of stop solutionwas added in tubes (not for blank), the contents were mixed and allowedto stand for 30 minutes at room temperature. Tubes were centrifuged 4000rpm for 10 minutes (Hettich Rotanta 460). One ml of clear supernatantwas mixed with 2.5 ml 0.5 M Na₂CO₃ and 0.5 ml diluted Folin reagent.After waiting for 10 mM (color development) the absorbance of themixture (color) was measured at 660 nm against an enzyme blank.

At least two parallel samples were used in each measurement.

Example 4 Purification of the Recombinant Protease

Cells and solids were removed from the spent culture medium obtainedfrom the fermentation (Example 2) by centrifugation for 30 min, 50000 gat +4° C. (Sorvall RC6 plus). 8 ml of the supernatant was used forpurification of protease. All purification steps were performed at coldroom. After centrifugation, sample was filtered through 0.44 filter(MILLEX HV Millipore) before applying to HiPrep 26/10 Desalting column(from GE Healthcare) equilibrated in 20 mM MES pH 5.3. Gel filteredsample was applied to a 1 mL S Sepharose HP column (from GE Healthcare)equilibrated in 20 mM MES pH 5.3.

Proteins were eluted using increasing NaCl gradient (0.5 M). Proteasecontains fractions were pooled and concentrated using Amicon Ultra-410,000 CO Centrifugal filter devices MILLIPORE. Sample was furtherpurified using Superdex 75 gel filtration column equilibrated with 20 mMMES, 150 mM NaCl pH 5.3. Protease contains fractions were combined andused for characterization of pH and temperature profiles. Final samplewas analysed on SDS PAGE gel FIG. 4.

Example 5 Characterisation of the Recombinant Protease TemperatureProfile

Temperature profile was obtained for the recombinant Malbrancheaprotease and Savinase® 16 L by using the assay described in Example 3.The result is shown in FIG. 5A. The protease has a temperature optimumaround 70° C.

pH-profile

The pH profile of the Malbranchea protease and Savinase® 16 L weredetermined at 50° C. using casein as a substrate as described in Example3, except that enzyme samples were diluted and casein was dissolved in40 mM Britton-Robinson buffer, the pH of the reaction was adjusted to pH6-10, the reaction time was 30 min and the enzyme reactions were stoppedusing a 0.11 M TCA solution which contained 0.22 M sodium acetate and0.33 M acetic acid. The results are shown in FIG. 5B. The recombinantprotease exhibits relative activity over 50% from pH 6 to pH 10 withbest activity around pH 10.

Example 6 Stain Removal Performance of Malbranchea ALKO4122 Proteasewith Liquid Detergent at Different Temperatures

Malbranchea ALKO4122 protease produced in Trichoderma, as described inExample 2 (b) was tested for its ability to remove blood/milk/inkstandard stain at 10, 20, 30 and 50° C. in the presence of Commercialliquid detergent (Table 3), at concentration of 5 g/1.

Standard stain, artificially soiled test cloth Art. 117 (blood/milk/ink,polyester+cotton, Serial No 11-08 or 10-07, EMPA Testmaterialen AG,Switzerland) was used as test material. Commercial protease preparationsSavinase® 16 L and Savinase® Ultra 16 L (contains a protease inhibitor,4-FBPA) and treatment without enzyme (control) were used for comparison.Each enzyme preparation was dosed 0-8 or 0-16 activity units (μgtyrosine/min) per ml wash liquor. Activity was measured as described inExample 3.

TABLE 3 Composition of Commercial liquid detergent. Ingredient % Anionicsurfactants 15-30 Nonionic surfactants, soap  5-15 Phosphonate,polycarboxylate 5 Optical brighteners and perfumes pH 8.2-8.6

5 g of Commercial liquid detergent was dissolved in 1 liter of tap water(dH≦4), mixed well with magnetic stirrer and tempered to washingtemperature. The pH in the wash liquor was approx. 8. The stain fabricwas first cut in to 1.5 cm×1.5 cm swatches and the pieces were maderounder by cutting the corners. Pieces were placed in wells ofmicrotiter plates (Nunc 150200). Into each well having a diameter of 2cm, 1.5 ml wash liquor containing detergent and enzyme dilution in water(approx. 50 μl) was added on top of the fabric. The plates with sampleswere in incubated in Infors Ecotron incubator shaker at 10, 20, 30 and50° C. for 60 min with 130 rpm. After that the swatches were carefullyrinsed under running water (appr. at washing temperature) and driedovernight at indoor air, on a grid, protected against daylight.

The stain removal effect was evaluated by measuring the colour asreflectance values with Minolta CM 2500 spectrophotometer using L*a*b*colour space coordinates (illuminant D65/2°). The colour from both sidesof the swatches was measured after the treatment. Each value was theaverage of at least 2 parallel fabric samples measured from both side ofthe fabric. Fading of blood/milk/ink stain, indicating the proteaseperformance (stain removal efficiency), was calculated as ΔL* (deltaL*), which means lightness value L* of enzyme treated fabric minuslightness value L* of fabric treated with washing liquor without enzyme(enzyme blank, control).

The results shown in FIGS. 6A-6D indicate that Malbranchea ALKO4122protease has considerably better effect on blood/milk/ink stain (Art.117, Serial No 11-08) at all temperatures tested compared to commercialSavinase® preparations. It was noticed that older batch (Serial No10-07) of blood/milk/ink stain was more difficult to remove withoutenzyme (with detergent only), and therefore the enzyme contribution(ΔL*) was significantly higher compared to tests performed with newerbatch 11-08. Malbranchea preparation of was even more efficient on thisdifficult old stain material compared to Savinase® (FIGS. 7A-7D). Alsoif dosing is calculated as amount of added protein (FIGS. 8A-8D), thestain removal efficiency was highest with Malbranchea sulfurea ALKO4122protease. The amount of protein from the enzyme preparations wasdetermined by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules,Calif.) using bovine gammaglobulin (Bio-Rad) as standard.

It is surprising that Malbranchea ALKO4122 protease shows optimal stainremoval performance at very broad temperature range and especially atlow temperatures, like 10-30° C., despite of its high temperatureoptimum at analytical conditions on casein substrate (approx. 70° C.,FIG. 5A). Savinase® that has similar temperature profile in analyticalconditions (FIG. 5A) shows clearly lower performance at cold washingtemperatures. The results of these tests indicate that MalbrancheaALKO4122 protease has excellent performance with liquid detergent atbroad temperature range and even at very cold washing temperatures.

Example 7 Launder Tests of Malbranchea ALKO 4122 Protease with LiquidDetergent and Different Stains

Malbranchea ALKO4122 protease produced in Trichoderma, as described inExample 2 (b), was tested for its ability to remove different stainswith Commercial liquid detergent at 30 and 60° C. compared to commercialprotease preparation Savinase® Ultra 16 L. The following artificiallysoiled test cloths from EMPA were used: blood/milk/ink (Art. 117, SerialNo 11-08 and 10-07, polyester+cotton), blood/milk/ink (Art. 116, SerialNo. 18-16, cotton), grass (Art. 164, Serial No. 23-03, cotton) and cocoa(Art. 112, Serial No. 31-06, cotton). The fabric was cut in 6 cm×6 cmswatches and the edges were neated by zig-zag stitches.

Stain removal treatments were performed in LP-2 Launder Ometer asfollows. Launder Ometer was first preheated to 30 or 60° C. Then 0, 2,5, 10 and 20 (40) activity units of enzyme per ml wash liquor was addedinto 1.2 liter containers containing 250 ml of tempered wash liquor andthe stain swatches. Activity (μg tyrosine/min) was measured as describedin Example 3. The wash liquor contained 5 g Commercial liquid detergent(Table 3) per litre tap water (dH≦4) and its pH was approx. 8. TheLaunder Ometer was run at 30° C. for 60 min with a rotation speed of 42rpm. After that the swatches were carefully rinsed under running water(ca. 20° C.) and dried overnight at indoor air, on a grid, protectedagainst daylight.

The colour of the swatches after treatment was measured with Minolta CM2500 spectrophotometer using L*a*b* color space coordinates and stainremoval effect calculated as ΔL* as described in Example 6. The colourfrom both sides of the swatches was measured after the treatment. Eachvalue was the average of at least 12 measurements per swatch. Themeasurements were avoided from areas with crease marks formed during thetreatment because of the folding of the fabric (in cotton stains Art.116 and Art. 112).

The results shown in FIGS. 9A-9H indicate that Malbranchea sulfureaALKO4122 protease has better effect on grass stain (Art. 164, Serial No.23-03) and different blood/milk/ink stains (Art. 117, Serial No 11-08and 10-07, Art. 116, Serial No 18-16) both at 30 and 60° C. compared tocommercial protease preparation Savinase® Ultra16 L. The dosage of 10-20units of Savinase® Ultra 16 L per was liquor was equal to dosage ofapproximately 0.4-0.7% of enzyme preparation per weight of detergent,which is in typical use level range for detergent enzymes.

Also results obtained with cocoa stain were better with Malbrancheacompared to Savinase® Ultra 16 L (data not shown). In additional Laundertests (data not shown) Malbranchea protease was found to be effective at30° C. also on the following stains of CFT (Center for Testmaterials BV,The Netherlands: Groundnutoil, pigments, high milk (C-10), Egg yolk,pigment, aged (CS-38), Grass extract (CS-08)). Same commercial liquiddetergent was used in all the tests. Results of these tests indicatethat Malbranchea protease is efficient on several stains at broadtemperature range 30-60° C.

Example 8 Stain Removal Performance of Malbranchea ALKO4122 Proteasewith Detergent Powders

The ability of Malbranchea ALKO4122 protease produced in Trichoderma toremove blood/milk/ink standard stain in the presence of detergent (5g/l) was tested at 50° C. using similar test system as described inExample 7, except Commercial traditional detergent powder containingbleaching agents, optical brighteners and phosphates/phosphonates andECE reference detergent 77 without optical brightener and bleachingagents (Art. 601, EMPA) were used. Also the colour of the swatches aftertreatment was measured with Minolta CM 2500 spectrophotometer usingL*a*b* colour space coordinates and stain removal effect calculated asΔL* as described in Example 6.

Results (FIGS. 10A and 10B) show that Malbranchea ALKO4122 protease canalso be used with detergent powders at highly alkaline conditions (pHapprox. 10-10.5).

Example 9 Storage Stability of Malbranchea ALKO4122 Protease

Storage of Malbranchea ALKO4122 protease produced in Trichoderma at 37°C. was tested using the following formulation recipes: 1) 0.2% (w/w)Proxel LV (preservative, Arch Biosides, U.K), pH adjusted to 6, 2) 20%propylene glycol, pH adjusted 6, 3) 50% propylene glycol, pH adjusted5.5. The samples of were packed in Sarstedt's test tubes (13 ml) andincubated at 37° C. Activity was measured (as described in Example 3) atcertain intervals and compared to earlier results obtained with Fusariumprotease Fe_RF6318 (WO2010125174A1) at similar conditions.

FIG. 11 shows that Malbranchea ALKO4122 protease has excellent storagestability at elevated temperatures (37° C.) compared to wild typeFusarium protease Fe_RF6318. When propylene glycol was used forstabilization, the residual activity of Fusarium protease was about 20%after 7 days incubation, whereas Malbranchea protease had not lostactivity in that time.

Example 10 Stability of Malbranchea ALKO4122 Protease in LiquidDetergents

Stability of Malbranchea ALKO4122 protease produced in Trichoderma at37° C. was tested in Commercial liquid detergent (Table 3) and EcolabelReference Detergent, light duty (Ch. Nr. 196-391, wfk Testgewebe GmbH).Savinase® 16 L and Fusarium protease Fe_RF6318 (WO2010125174A1) wereused as references.

The following test systems were used: 0.4 g enzyme preparation and 9.6 gof detergent solution were mixed well in Sarstedt's test tubes (13 ml).0.2% of Proxel LV was added as preservative in detergent before mixingit with other components. The pH value of the samples prepared inCommercial liquid detergent was approx. 8. pHs of the samples preparedin Ecolabel Reference Detergent were approx. 7.2. Test tubes wereincubated at 37° C. and the protease activity was measured at certainintervals according to the method described in Example 3.

FIG. 12 shows that Malbranchea ALKO4122 has very good storage stabilityat elevated temperatures (37° C.) in liquid detergents compared to wildtype Fusarium protease Fe_RF6318 (WO2010125174A1). Malbranchea proteasehad similar stability compared to Savinase® 16 L in Ecolabel ReferenceDetergent.

At room temperature an excellent stability was observed (data notshown).

REFERENCES

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1. A serine protease enzyme, wherein said enzyme has serine proteaseactivity and comprises an amino acid sequence having at least 66%identity to the amino acid sequence set forth in SEQ ID NO:18.
 2. Theserine protease enzyme of claim 1, wherein said enzyme comprises anamino acid sequence having at least 85% identity to the amino acidsequence set forth in SEQ ID NO:18.
 3. The serine protease enzyme ofclaim 1, wherein said enzyme comprises an amino acid sequence set forthin SEQ ID NO:18.
 4. The serine protease enzyme of claim 1, wherein saidenzyme consists of an amino acid sequence set forth in SEQ ID NO:18. 5.The serine protease enzyme of claim 1, wherein said enzyme is obtainedfrom Malbranchea ALKO4122 deposited strain CBS 128533 or fromMalbranchea ALKO4178 deposited strain CBS
 128564. 6. The serine proteaseenzyme of claim 1, wherein a mature form of said enzyme has a molecularmass between 20 and 35 kDa.
 7. The serine protease enzyme of claim 1,wherein said enzyme has a temperature optimum at pH 8.5 using 30 minreaction time and casein as a substrate from 30° C. to 80° C.
 8. Theserine protease of claim 1, wherein said enzyme has pH optimum at 50° C.temperature using 30 min reaction time and casein as a substrate from atleast pH 6 to pH
 10. 9. The serine protease enzyme of claim 1, whereinsaid enzyme degrades or removes proteinaceous stains in the presence ofdetergent at a temperature between 0 and 90° C.
 10. An isolated nucleicacid molecule comprising a polynucleotide sequence which encodes aserine protease enzyme selected from the group consisting of: (a) anucleic acid molecule encoding a polypeptide having serine proteaseactivity and comprising the amino acid sequence set forth in SEQ IDNO:18; (b) a nucleic acid molecule encoding a polypeptide having serineprotease activity and at least 66% identity to the amino acid sequenceset forth in SEQ ID NO:18; (c) a nucleic acid molecule comprising thecoding sequence of the nucleotide sequence as set forth in SEQ ID NO:17;(d) a nucleic acid molecule comprising the coding sequence of thepolynucleotide sequence contained in DSM 24410; (e) a nucleic acidmolecule the coding sequence of which differs from the coding sequenceof a nucleic acid molecule of any one of (c) to (d) due to thedegeneracy of the genetic code; and (g) a nucleic acid moleculecomprising a polynucleotide sequence which hybridizes under stringentconditions to a nucleic acid molecule contained in DSM 24426, or setforth in SEQ ID NO:17 encoding a polypeptide having serine proteaseactivity and an amino acid sequence which shows at least 66% identity tothe amino acid sequence as set forth in SEQ ID NO:18.
 11. The isolatednucleic acid molecule of claim 10, wherein said nucleic acid moleculecomprises a polynucleotide sequence that hybridizes under stringentconditions to (i) a polynucleotide sequence comprising the nucleotidesequence set forth in SEQ ID No:11; or (ii) a polynucleotide sequencecomprising the nucleotide sequence set forth in SEQ ID No:12.
 12. Arecombinant expression vector comprising the nucleotide sequence ofclaim 10 operably linked to regulatory sequences capable of directingexpression of said serine protease enzyme in a suitable host.
 13. A hostcell comprising the recombinant expression vector of claim
 12. 14. Thehost cell of claim 13, wherein said host is a microbial host.
 15. Thehost cell of claim 13, wherein said host is a filamentous fungus. 16.The host cell of claim 13, wherein said host is of a genus Trichoderma,Aspergillus, Fusarium, Humicola, Chrysosporium, Neurospora, Rhizopus,Penicillium, Myceliophthora and Mortiriella.
 17. The host cell of claim16, wherein said host is Trichoderma or Aspergillus.
 18. The host cellof claim 17, wherein said host is T. reesei.
 19. A process of producinga polypeptide having serine protease activity, said process comprisingthe steps of culturing the host cell of claim 13 and recovering thepolypeptide.
 20. A polypeptide having serine protease activity encodedby the nucleic acid sequence of claim 10 and which is obtained by theprocess of claim
 19. 21. A process for obtaining an enzyme preparationcomprising the steps of culturing a host cell of claim 13 and eitherrecovering the polypeptide from the cells or separating the cells fromthe culture medium and obtaining the supernatant.
 22. An enzymepreparation obtainable by the process of claim
 21. 23. An enzymepreparation, which comprises the serine protease enzyme of claim
 1. 24.The enzyme preparation of claim 22 wherein said preparation comprisesother enzymes selected from the group consisting of proteases, amylases,cellulases, lipases, xylanases, mannanases, cutinases, pectinases andoxidases, with or without a mediator.
 25. The enzyme preparation ofclaim 22 wherein said preparation comprises one or more additivesselected from the group consisting of stabilizers, buffers, surfactants,builders, bleaching agents, mediators, anti-corrosion agents,antiredeposition agents, caustics, abrasives, optical brighteners, dyes,perfumes, pigments, and preservatives.
 26. The enzyme preparation ofclaim 22, wherein said enzyme preparation is in the form of liquid,powder, granulate or tablets.
 27. Use of the serine protease enzyme ofclaim 1 for detergents, for treating fibers, for treating proteinaceousmaterials selected preferably from wool, hair, leather and silk, fortreating food or feed, or for applications involving modification,degradation or removal of proteinaceous material.
 28. Use of the serineprotease enzyme of claim 1 as a detergent additive, preferably in liquiddetergents, in detergent powders and in detergent tablets.
 29. Adetergent composition, wherein said composition comprises the serineprotease enzyme of claim 1 and additives selected from the groupconsisting of stabilizers, buffers, surfactants, builders, bleachingagents, mediators, anti-corrosion agents, antiredeposition agents,caustics, abrasives, optical brighteners, dyes, perfumes, pigments, andpreservatives.
 30. The detergent composition of claim 29, wherein saidcomposition comprises other enzymes selected from the group consistingof proteases, amylases, cellulases, lipases, xylanases, mannanases,cutinases, pectinases and oxidases, with or without a mediator.
 31. Amethod for treating a proteinaceous material selected from the groupconsisting of wool, hair, leather, silk, food, and feed, the methodcomprising contacting the proteinaceous material with the serineprotease enzyme of claim 1 for a sufficient time to achieve at leastpartial degradation of the proteinaceous material.
 32. A method fordegrading or removing proteinaceous material from the surface of solidwater-insoluble carrier, the method comprising contacting theproteinaceous material with the serine protease enzyme of claim 1 for asufficient time to achieve at least partial degradation of theproteinaceous material.
 33. A method of preparing a detergent,comprising adding the serine protease enzyme of claim 1 as a detergentadditive to a detergent selected from the group consisting of liquiddetergents, detergent powders, and detergent tablets.